Abstract

scVEGF/(177)Lu is a novel radiopharmaceutical targeted by recombinant single-chain (sc) derivative of vascular endothelial growth factor (VEGF) that binds to and is internalized by vascular endothelial growth factor receptors (VEGFR). scVEGF/(177)Lu potential as adjuvant and neoadjuvant anti-angiogenic therapy was assessed in metastatic and orthotopic mouse models of triple-negative breast cancer.Metastatic lesions in Balb/c mice were established by intracardiac injection of luciferase-expressing 4T1luc mouse breast carcinoma cells. Mice with metastatic lesions received single intravenous (i.v.) injection of well-tolerated dose of scVEGF/(177)Lu (7.4 MBq/mouse) at day 8 after 4T1luc cell injection. Primary orthotopic breast tumors in immunodeficient mice were established by injecting luciferase-expressing MDA231luc human breast carcinoma cells into mammary fat pad. Tumor-bearing mice were treated with single injections of scVEGF/(177)Lu (7.4 MBq/mouse, i.v), or liposomal doxorubicin (Doxil, 1 mg doxorubicin per kg, i.v.), or with a combination of Doxil and scVEGF/(177)Lu given at the same doses, but two hours apart. "Cold" scVEGF-targeting conjugate was included in controls and in Doxil alone group. The effects of treatments were defined by bioluminescent imaging (BLI), computed tomography (CT), computed microtomography (microCT), measurements of primary tumor growth, and immunohistochemical analysis.In metastatic model, adjuvant treatment with scVEGF/(177)Lu decreased overall metastatic burden and improved survival. In orthotopic primary tumor model, a combination of Doxil and scVEGF/(177)Lu was more efficient in tumor growth inhibition than each treatment alone. scVEGF/(177)Lu treatment decreased immunostaining for VEGFR-1, VEGFR-2, and pro-tumorigenic M2-type macrophage marker CD206.Selective targeting of VEGFR with well-tolerated doses of scVEGF/(177)Lu is effective in metastatic and primary breast cancer models and can be combined with chemotherapy. As high level of VEGFR expression is a common feature in a variety of cancers, targeted delivery of (177)Lu for specific receptor-mediated uptake warrants further exploration.

Abstract

Here, we evaluate [(99m)Tc]annexin V-128, an in vivo marker of apoptosis, for single photon emission computed tomography (SPECT) imaging of localization and antibiotic treatment of disseminated bacterial infection, using a well-described mouse model that employs bioluminescent Listeria monocytogenes.Sixteen groups of five mice in six separate experiments were infected with bioluminescent Listeria, and in vivo bioluminescence imaging (BLI) was performed each day, to assess the location and extent of infection and response to antibiotics. [(99m)Tc]annexin V-128 was then injected for SPECT imaging, and the two sets of images were correlated and validated.Signals from BLI and [(99m)Tc]annexin V-128 SPECT co-localized within the spleen and other organs including bone marrow, intestine, nasopharynx, and brain. Decreases in [(99m)Tc]annexin V-128 uptake and BLI signal within the spleen directly reflected the reduction of bacterial infection by ampicillin treatment.Tc-99m-Annexin V-128 uptake as observed by SPECT allowed for the detection of systemic listeriosis and ampicillin treatment in mice. [(99m)Tc]annexin V-128 should be further explored for the assessment of bacterial spread and antibiotic efficacy in patients with disseminated bacterial infection.

Abstract

Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome.Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs.Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome.

Abstract

The brain demonstrates spontaneous low-frequency (<0.1Hz) cerebral blood flow (CBF) fluctuations, measurable by resting state functional MRI (rs-fMRI). Ultra small superparamagnetic iron oxide particles have been shown to enhance task-based fMRI signals (cerebral blood volume fMRI or CBV-fMRI), compared to the BOLD effect, by a factor of ?2.5 at 3T in primates and humans. We evaluated the use of ferumoxytol for steady state, resting state FMRI (CBV-rs-fMRI) and relative cerebral blood volume (rCBV) mapping, at 3T, in healthy volunteers. All standard resting state networks (RSNs) were identified in all subjects. On average the RSN Z statistics (MELODIC independent components) and volumes of the visual and default mode (DMN) networks were comparable. rCBV values were averaged for the visual (Vis) and DMN networks and correlated with the corresponding DMN and visual network Z statistics. There was a negative correlation between the rCBV and the Z statistics for the DMN, for both BOLD and CBV-rs-fMRI contrast (R(2)=0.63, 0.76). A similar correlation was not found for the visual network. Short repetition time rs-fMRI data were Fourier transformed to evaluate the effect of ferumoxytol on cardiac and respiratory fluctuations in the brain rs-BOLD, CBV signals. Cardiac and respiratory fluctuations decreased to baseline within large vessels post ferumoxytol. Robust rs-fMRI and CBV mapping is possible in normal human brain.

Abstract

There is much that can be done to detect apoptosis and other forms of cell death with existing clinical modalities including ultrasound, MRI, and optical imaging without the need for current or new intravenous contrast agents. We will discuss how these widely available imaging technologies can readily be applied to the imaging of apoptosis in patients undergoing chemotherapy or radiation treatment. The limiting factor of course is the lack of knowledge of the optimal times after the start of treatment for the most accurate assessment of apoptosis and necrosis with each modality and specific technique. It is hoped that imaging studies that systematically look at treatment response can soon be performed to address these issues.

Abstract

While decreased ATP production and redox imbalance are central to mitochondrial disease pathogenesis, efforts to develop effective treatments have been hampered by the lack of imaging markers of oxidative stress. In this study we wished to determine if Tc99m-HMPAO, a SPECT imaging marker of cerebral blood flow and glutathione/protein thiol content, could be used to monitor the effect(s) of EPI-743, an oral redox modulating, para-benzoquinone based therapeutic for mitochondrial disease. We hypothesized that treatment changes in HMPAO uptake would be inversely proportional to changes in oxidative stress within the brain and directly correlate to clinical response to EPI-743 therapy. Twenty-two patients with mitochondrial disease were treated with EPI-743. Each underwent baseline and 3-month Tc99m-HMPAO SPECT scanning along with clinical/neurologic evaluations. Diseases treated were: Leigh syndrome (n=7), polymerase ? deficiency (n=5), MELAS (n=5), Friedreich ataxia (n=2), Kearns-Sayre syndrome, Pearson syndrome, and mtDNA depletion syndrome. Neuro-anatomic uptake analyses of HMPAO were performed with NeuroGam? (Segami Corp.) statistical software and clinical response was assessed by the Newcastle Paediatric Mitochondrial Disease Scale or Newcastle Mitochondrial Disease Adult Scale depending on patient age. For all 22 patients there was a significant linear correlation between the change in cerebellar uptake of HMPAO and the improvement in Newcastle score (r=0.623, **p=0.00161). The MELAS subgroup showed a significant relationship of whole brain uptake (n=5, r=0.917, *p=0.028) to improvement in Newcastle score. We conclude that Tc99m-HMPAO SPECT scanning has promise as a general marker of the oxidative state of the brain and its response to redox modulating therapies. Further studies will be needed to confirm these findings in a more homogenous study population.

Abstract

In humans, apoptosis (programmed cell death) is the most common form of cell death after necrosis. Apoptosis is a series of genetically preprogrammed biochemical and morphologic energy-requiring events that, after a specific external or internal stimulus, results in the physiologic disappearance of a cell via its self-disintegration and packaging of its contents into membrane vesicles called apoptotic bodies. Apoptotic bodies can readily be ingested, with their nutrients and even organelles recycled by neighboring cells or phagocytes without local inflammation. In contrast, necrosis is characterized by the primary loss of plasma membrane integrity and the uncontrolled release of a cell's contents, often causing local inflammation, tissue damage, and scarring. Alternate forms of cell death also exist, associated with specific molecular mechanisms involving enzymes, organelles, genes, external stimuli, or blockade of normal cell proliferation. In this review we will briefly outline the molecular mechanisms of apoptosis that can be imaged with radiotracers now under development.

Abstract

Inherited mitochondrial respiratory chain disorders are progressive, life-threatening conditions for which there are limited supportive treatment options and no approved drugs. Because of this unmet medical need, as well as the implication of mitochondrial dysfunction as a contributor to more common age-related and neurodegenerative disorders, mitochondrial diseases represent an important therapeutic target. Thirteen children and one adult with genetically-confirmed mitochondrial disease (polymerase ? deficiency, n=4; Leigh syndrome, n=4; MELAS, n=3; mtDNA deletion syndrome, n=2; Friedreich ataxia, n=1) at risk for progressing to end-of-life care within 90 days were treated with EPI-743, a novel para-benzoquinone therapeutic, in a subject controlled, open-label study. Serial measures of safety and efficacy were obtained that included biochemical, neurological, quality-of-life, and brain redox assessments using technetium-99m-hexamethylpropyleneamine oxime (HMPAO) single photon emission computed tomography (SPECT) radionuclide imaging. Twelve patients treated with EPI-743 have survived; one polymerase ? deficiency patient died after developing pneumonia and one patient with Surf-1 deficiency died after completion of the protocol. Of the 12 survivors, 11 demonstrated clinical improvement, with 3 showing partial relapse, and 10 of the survivors also had an improvement in quality-of-life scores at the end of the 13-week emergency treatment protocol. HMPAO SPECT scans correlated with clinical response; increased regional and whole brain HMPAO uptake was noted in the clinical responders and the one subject who did not respond clinically had decreased regional and whole brain HMPAO uptake. EPI-743 has modified disease progression in >90% of patients in this open-label study as assessed by clinical, quality-of-life, and non-invasive brain imaging parameters. Data obtained herein suggest that EPI-743 may represent a new drug for the treatment of inherited mitochondrial respiratory chain disorders. Prospective controlled trials will be undertaken to substantiate these initial promising observations. Furthermore, HMPAO SPECT imaging may be a valuable tool for the detection of central nervous system redox defects and for monitoring response to treatments directed at modulating abnormal redox.

Abstract

Tumor vessels abundantly express receptors for vascular endothelial growth factor (VEGF), despite treatment with conventional or antiangiogenic drugs. We wished to determine whether the high levels of VEGF receptor (VEGFR) within the tumor vasculature could be leveraged for intracellular delivery of therapeutically significant doses of scVEGF/(177)Lu, a novel radiopharmaceutical based on a recombinant single-chain (sc) derivative of VEGF, in orthotopic breast cancer models.scVEGF-PEG (polyethylene gycol)-DOTA conjugates containing 2.0-, 3.4-, or 5.0-kDa PEG linkers site-specifically conjugated to a cysteine-containing tag (Cys-tag) in scVEGF were radiolabeled with (177)Lu (scVEGF/(177)Lu) for in vivo studies. Human MDA231luc and mouse 4T1luc cell lines were injected orthotopically to establish breast carcinoma tumors in immunodeficient and immunocompetent hosts, respectively. The effects of scVEGF/(177)Lu were defined by analysis of changes in tumor growth and immunohistochemical staining for the endothelial markers CD31 and VEGFR-2 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining for intratumoral apoptosis.Biodistribution assays and dosimetric calculations established that scVEGF/(177)Lu with a 3.4-kDa PEG linker delivered the highest dose of radiation to tumors (69.9 cGy/MBq/g of tissue) and the lowest dose to the kidneys (33.3 cGy/MBq/organ). Total doses below 40 MBq/mouse of scVEGF/(177)Lu did not affect renal function, and 3 divided doses of 6.3 MBq/mouse or a bolus dose of 18.9 MBq/mouse induced only transient lymphopenia and weight loss (<10% baseline weight). In mice with orthotopic mammary breast carcinoma, intravenous injections of well-tolerated bolus and fractionated doses of scVEGF/(177)Lu in the range from 6.3 to 18.9 MBq/mouse (25-76 MBq/m(2)) resulted in dose-dependent tumor growth inhibition. Immunohistochemical analysis of tumors at 4-5 wk after single injections of scVEGF/(177)Lu indicated dose-dependent regression of tumor vasculature and widespread intratumoral apoptosis. A single dose of 7.4 MBq/mouse of scVEGF/(177)Lu given before a course of bevacizumab or sunitinib treatment enhanced the antiangiogenic effects of both drugs.Selective targeting of VEGFR in tumor vasculature with well-tolerated doses of scVEGF/(177)Lu is effective in orthotopic breast cancer models. As high levels of VEGFR expression in the tumor vasculature are a common feature in a variety of cancers, targeting tumor angiogenesis with scVEGF/(177)Lu warrants further exploration.

Abstract

The purposes of this review are to describe the signaling pathways of and the cellular changes that occur with apoptosis and other forms of cell death, summarize tracers and modalities used for imaging of apoptosis, delineate the relation between apoptosis and inhibition of protein translation, and describe spectroscopic technologies that entail high-frequency ultrasound and infrared and midinfrared light in characterizing the intracellular events of apoptosis.Apoptosis is a highly orchestrated set of biochemical and morphologic cellular events. These events present many potential targets for the imaging of apoptosis in vivo. Imaging of apoptosis can facilitate early assessment of anticancer treatment before tumor shrinkage, which may increase the effectiveness of delivery of chemotherapy and radiation therapy and speed drug development.

Abstract

We developed a recombinant form of human annexin VI called annexin VI-601 (M(r) 76,224) with the N-terminal extension of Ala-Gly-Gly-Cys-Gly-His to allow ready attachment of fluorescent or radioactive labels. The protein was produced by expression in E. coli and was purified by calcium-dependent membrane binding, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. The protein could be readily labeled with iodoacetamidofluorescein and with (99m)Tc. The protein bound with high affinity to PS-containing phospholipid vesicles and to erythrocytes with exposed phosphatidylserine. Fluorescent annexin VI-601 readily detected apoptosis of Jurkat cells by flow cytometry at much lower calcium concentrations than those required for equivalent detection by annexin V. In vivo administration of radiolabeled protein showed that blood clearance was much slower than annexin V. In conclusion, annexin VI may have advantages over annexin V in certain situations for both in vitro and in vivo detection of apoptosis and therapeutic targeting of PS due to its lower calcium requirement for membrane binding and its higher molecular weight.

Abstract

Several drugs targeting vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are approved for cancer treatment. However, these drugs induce relatively modest and frequently unpredictable tumor responses. In this work, we explored whether noninvasive imaging of VEGFR, a direct target of antiangiogenic drugs, can provide real-time information on responses to the treatment with sunitinib, a small-molecule VEGFR inhibitor approved by the Food and Drug Administration.We imaged VEGFR in an orthotopic mammary tumor model during the course of treatment with sunitinib using a recently developed SPECT tracer, a (99m)Tc-labeled single-chain VEGF (scVEGF), that binds to and is internalized by VEGFR. Tumors from imaged mice were harvested and cryosectioned, and alternating sections were analyzed by autoradiography and immunohistochemistry to determine the expression of endothelial cell markers VEGFR-2 and CD31.In vitro assays with endothelial cells overexpressing VEGFR-2 established that sunitinib does not inhibit VEGFR-2-mediated uptake of scVEGF-based tracers. SPECT and autoradiography with (99m)Tc-scVEGF of tumor cryosections revealed a 2.2- to 2.6-fold decrease in tracer uptake after 4 daily doses of sunitinib. However, once treatment was discontinued, tracer uptake rapidly (3 d) increased, particularly at the tumor edges. Immunohistochemical analysis of VEGFR-2 and CD31 supported SPECT and autoradiographic imaging findings, revealing the corresponding depletion of VEGFR-2- and CD31-positive endothelial cells from tumor vasculature during therapy and the rapid reemergence of VEGFR-2- and CD31-positive vasculature at the tumor edges after discontinuation of treatment.Our findings suggest that imaging with (99m)Tc-scVEGF might be useful for monitoring VEGFR responses to antiangiogenic treatment regimens.

Abstract

Ischemic insult to the myocardium is associated with cardiomyocyte apoptosis. Because apoptotic cell death is characterized by phosphatidylserine externalization on cell membrane and annexin-A5 (AA5) avidly binds to phosphatidylserine, we hypothesized that radiolabeled AA5 should be able to identify the regions of myocardial ischemia.Models of brief myocardial ischemia by the occlusion of the coronary artery for 10 min (I-10) and reperfusion for 180 min (R-180) for the detection of phosphatidylserine exteriorization using (99m)Tc-labeled AA5 and gamma-imaging were produced in rabbits. (99m)Tc-AA5 uptake after brief ischemia was compared with an I-40/R-180 infarct model. Histologic characterization of both myocardial necrosis and apoptosis was performed in ischemia and infarct models. Phosphatidylserine exteriorization was also studied in a mouse model, and the dynamics and kinetics of phosphatidylserine exposure were assessed using unlabeled recombinant AA5 and AA5 labeled with biotin, Oregon Green, or Alexa 568. Appropriate controls were established.Phosphatidylserine exposure after ischemia in the rabbit heart could be detected by radionuclide imaging with (99m)Tc-AA5. Pathologic characterization of the explanted rabbit hearts did not show apoptosis or necrosis. Homogenization and ultracentrifugation of the ischemic myocardial tissue from rabbit hearts recovered two thirds of the radiolabeled AA5 from the cytoplasmic compartment. Murine experiments demonstrated that the cardiomyocytes expressed phosphatidylserine on their cell surface after an ischemic insult of 5 min. Phosphatidylserine exposure occurred continuously for at least 6 h after solitary ischemic insult. AA5 targeted the exposed phosphatidylserine on cardiomyocytes; AA5 was internalized into cytoplasmic vesicles within 10-30 min. Twenty-four hours after ischemia, cardiomyocytes with internalized AA5 had restored phosphatidylserine asymmetry of the sarcolemma, and no detectable phosphatidylserine remained on the cell surface. The preadministration of a pan-caspase inhibitor, zVAD-fmk, prevented phosphatidylserine exposure after ischemia.After a single episode of ischemia, cardiomyocytes express phosphatidylserine, which is amenable to targeting by AA5, for at least 6 h. Phosphatidylserine exposure is transient and internalized in cytoplasmic vesicles after AA5 binding, indicating the reversibility of the apoptotic process.

Abstract

Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) drive angiogenesis, and several VEGFR inhibitors are already approved for use as single agents or in combination with chemotherapy. Although there is a clear benefit with these drugs in a variety of tumors, the clinical response varies markedly among individuals. Therefore, there is a need for an efficient method to identify patients who are likely to respond to antiangiogenic therapy and to monitor its effects over time. We have recently developed a molecular imaging tracer for imaging VEGFRs known as scVEGF/(99m)Tc; an engineered single-chain (sc) form of VEGF radiolabeled with technetium Tc 99m ((99m)Tc). After intravenous injection, scVEGF/(99m)Tc preferentially binds to and is internalized by VEGFRs expressed within tumor vasculature, providing information on prevalence of functionally active receptors. We now report that VEGFR imaging readily detects the effects of pazopanib, a small-molecule tyrosine kinase inhibitor under clinical development, which selectively targets VEGFR, PDGFR, and c-Kit in mice with HT29 tumor xenografts. Immunohistochemical analysis confirmed that the changes in VEGFR imaging reflect a dramatic pazopanib-induced decrease in the number of VEGFR-2(+)/CD31(+) endothelial cells (ECs) within the tumor vasculature followed by a relative increase in the number of ECs at the tumor edges. We suggest that VEGFR imaging can be used for the identification of patients that are responding to VEGFR-targeted therapies and for guidance in rational design, dosing, and schedules for combination regimens of antiangiogenic treatment.

Abstract

Angiogenesis plays a central role in the pathogenesis of chronic inflammatory disorders. Vascular endothelial growth factor (VEGF) and its receptors are the most important regulators of angiogenesis. We wished to determine whether labeled forms of single-chain VEGF (scVEGF) could be used to image VEGF receptors in a well-characterized model of sterile soft-tissue inflammation induced by intramuscular injection of turpentine.Anesthetized adult male Swiss-Webster mice received a 20-microL intramuscular injection of turpentine into the right thigh. At 4, 7, or 10 d later, groups of 3-5 mice were injected via the tail vein with 50 microg of either scVEGF that had been site specifically labeled with Cy5.5 (scVEGF/Cy) or inactivated scVEGF/Cy (inVEGF/Cy) and then examined by fluorescence imaging. At 3, 4, 6, 7, 9, 10, or 12 d, additional groups of 3-5 mice were injected via the tail vein with 74-111 MBq of (99m)Tc-scVEGF (or (99m)Tc-inVEGF) and then examined by SPECT imaging.On days 3 through 10, both forms of scVEGF (scVEGF/Cy and (99m)Tc-scVEGF) showed significantly higher uptake (P < 0.05) in the right (abscessed) thigh than in the contralateral thigh (and higher uptake than the inactivated tracer). Peak uptake occurred on day 7 (3.67 +/- 1.79 [ratio of uptake in abscessed thigh to uptake in normal thigh, mean +/- SD] and 0.72 +/- 0.01 for scVEGF/Cy and inVEGF/Cy, respectively, and 3.49 +/- 1.22 and 1.04 +/- 0.41 for (99m)Tc-scVEGF and (99m)Tc-inVEGF, respectively) and slowly decreased thereafter. Autoradiography revealed peak tracer uptake in the thick irregular angiogenic rim of the abscess cavity on day 9 (5.83 x 10(-7) +/- 9.22 x 10(-8) and 5.85 x 10(-8) +/- 5.95 x 10(-8) percentage injected dose per pixel for (99m)Tc-scVEGF and (99m)Tc-inVEGF, respectively); in comparison, a thin circumscribed rim of uptake was seen with (99m)Tc-inVEGF. Immunostaining revealed that VEGFR-2 (VEGF receptor) colocalized with CD31 (endothelial cell marker) at all time points in the abscess rim, whereas F4/80 (macrophage) immunostaining reached a maximum at day 7 and decreased by day 10.The uptake of scVEGF in turpentine-induced abscesses was specific and directly related to VEGFR-2 expression in the neovasculature of the angiogenic rim. Peak tracer uptake coincided with maximum macrophage infiltration, suggesting that scVEGF imaging may be useful for the detection, localization, and monitoring of chronic inflammation in bone, joints, or soft tissues.

Abstract

There is a rapid expansion in the number of new anti-cancer drugs with remarkably different mechanisms of action that can augment traditional chemotherapy. As these agents are often used in combination with traditional chemotherapy testing the effects of these novel agents has proven difficult requiring large sample sizes to detect relatively small differences in patient survival. Despite the wide variety of mechanisms, most new drugs are thought to ultimately induce apoptosis of tumor cells or their supportive vasculature. Imaging agents that can non-invasively monitor apoptosis in response to these new drugs could therefore help streamline the drug development process. They may also help guide oncologists to identify those patients that could best benefit from a given therapeutic regimen, dose, or duration of drug. In this article we will outline the existing imaging agents and modalities that are currently undergoing clinical testing and those that could be rapidly translated into humans.

Imaging the molecular signatures of apoptosis and injury with radiolabeled annexin V.Proceedings of the American Thoracic SocietyBlankenberg, F. G.2009; 6 (5): 469-476

Abstract

Annexin V is a ubiquitous intracellular protein in humans that has a variety of intriguing characteristics, including a nanomolar affinity for the membrane-bound constitutive anionic phospholipid known as phosphatidylserine (PS). PS is selectively expressed on the surface of apoptotic or physiologically stressed cells. As such, radiolabeled forms of annexin V have been used in both animal models and human Phase I and Phase II trials to determine if this tracer can be employed as an early surrogate marker of therapeutic efficacy in NSCLC and non-Hodgkin's lymphoma. Many other pulmonary imaging applications of radiolabeled annexin V are also possible, including the detection and monitoring of active pulmonary inflammation and other pathophysiologic stressors in a variety of diseases. In this article, the salient molecular features of apoptosis (and other forms of cell death) that permits imaging with radiolabeled annexin V will be discussed. The latest results from Phase II imaging trials with NSCLC and non-Hodgkin's lymphoma will be also be detailed. Finally, the potential future application of this tracer for the imaging of other pulmonary pathologies will be outlined.

Abstract

We describe a new generation of tracers for molecular imaging of the cell surface receptors for epidermal growth factor (EGF). These receptors play a key role in the progression of many tumors and are major drug development targets. Our tracers are based on a recombinant human EGF expressed with a cysteine-containing tag that enables facile site-specific radiolabeling with (99m)Tc for single photon emission computed tomography or site-specific conjugation of (64)Cu PEGylated chelators for positron emission tomography. These tracers retain EGF activities in vitro and display selective and highly specific focal uptake in tumors in vivo. We expect that nuclear imaging of EGF receptors with these tracers will be useful for clinical diagnosis, therapeutic monitoring, and development of new drugs and treatment regimens.

Abstract

The early assessment of a solid tumor's response to conventional or new drug therapy to complement or replace current RECIST (or other clinical) criteria remains an elusive goal. The work horse PET tracer (18)F-FDG, may represent the most immediate method to track individual tumor response to therapy for many types of cancer. Newer radiotracers such as radiolabeled annexin V, have also shown the ability to selectively localize to tumor cells undergoing apoptosis (programmed cell death) in response to successful treatment in vivo. In this article we will review therapy reduced tumor apoptosis and the radiotracers used to date to image this process in both animal models and clinical trials.

Abstract

Despite over a decade of intense investigation there is still no routine method for the clinical imaging of apoptosis in oncologic patients. There have been multiple tracers proposed but none as of yet has received FDA approval. Radiolabeled annexin V is one of the few radiotracers that has been widely used in Phase II trials and is still under development. In this review we will first detail the general mechanisms involved with apoptosis and other common forms of cell death. Next we will outline the latest in vivo imaging data in animal models and humans including that obtained with radiolabeled annexin V. It is hoped that improved understanding of the complex biochemical pathways involved with cell death will lead to at least several radiopharmaceuticals with the ability to image apoptosis as part of improving the care and treatment of patients suffering from cancer.

Abstract

After several decades of debate, it is now widely acknowledged that apoptosis, also known as programmed cell death, is central to homoeostasis and normal development and physiology in all multicellular organisms, including humans. The dysregulation of apoptosis can lead to the destruction of normal tissues in a variety of disorders, including autoimmune and neurodegenerative diseases (too much apoptosis) or the growth of tumors (too little apoptosis). In addition, effective therapy of tumors requires the iatrogenic induction of programmed cell death by radiation, chemotherapy, or both. Given the central role of apoptosis, it would be desirable to have a noninvasive imaging method to serially detect and monitor this process in cancer patients undergoing conventional radiation and chemotherapy treatments as well as for the development and testing of new drugs. In this article, the latest modalities and contrast agents described in the literature for the imaging of apoptosis in vivo are reviewed. First, the most recent developments in the biochemical characterization of the many intracellular pathways involved in this complex process are discussed. Next, a variety of new radionuclide tracers, including radiolabeled annexin V and caspase inhibitors for PET and SPECT, are described. Finally, the use of MRI, MR spectroscopy, and ultrasound as possible alternative imaging modalities for the imaging of apoptosis is addressed.

Abstract

We report that the pathologic components present within the atheromatous plaques of ApoE knock-out mice can reflect significant amounts of mid-infrared (mid-IR) light. Furthermore, the reflected light spectra contained the unique signatures of a variety of biologic features including those found in unstable or "vulnerable" plaque. This discovery may represent a unique opportunity to develop a new intravascular diagnostic modality that can detect and characterize sites of atherosclerosis.

Abstract

Angiogenesis is a fundamental feature of tumor development, and therefore, the tracers for molecular imaging of specific angiogenic biomarkers are expected to be useful for diagnostics, patient monitoring, and drug development. We have created a new class of imaging agents based on the most important mediator of angiogenesis, vascular endothelial growth factor (VEGF). Our latest version is a single-chain (sc) VEGF protein containing an N-terminal Cys-tag designed for site-specific modification with a variety of imaging and therapeutic moieties. We have recently found that the Cys-tag itself can form a stable chelate with (99m)Tc using tin-tricine as an exchange reagent. This self-chelation approach yields a highly stable and fully functional form of radiolabeled scVEGF that can be used as a SPECT tracer for tumor angiogenesis. Also of note is that directly labeled scVEGF has less than one-half the nonspecific renal uptake of (99m)Tc-HYNIC-scVEGF. The simple production of scVEGF for direct chelation of (99m)Tc makes it a promising molecular imaging agent for the oncology clinic.

Abstract

Since its original description in 1972, apoptosis or programmed cell death has been recognized as the major pathway by which the body precisely regulates the number and type of its cells as part of normal embryogenesis, development, and homeostasis. Later it was found that apoptosis was also involved in the pathogenesis of a number of human diseases, cell immunity, and the action of cytotoxotic drugs and radiation therapy in cancer treatment. As such, the imaging of apoptosis with noninvasive techniques such as with radiotracers, including annexin V and lipid proton magnetic resonance spectroscopy, may have a wide range of clinical utility in both the diagnosis and monitoring therapy of a wide range of human disorders. In this chapter we review the basic biochemical and morphologic features of apoptosis and the methods developed thus far to image this complex process in humans.

Abstract

Targeted delivery of therapeutic and imaging agents requires conjugation of a corresponding payload to a targeting peptide or protein. The ideal procedure should yield a uniform preparation of functionally active conjugates and be translatable for development of clinical products. We describe here our experience with site-specific protein modification via a novel cysteine-containing fusion tag (Cys-tag), which is a 15-amino-acid (aa) N-terminal fragment of human ribonuclease I with the R4C substitution. Several Cys-tagged proteins and peptides with different numbers of native cysteines were expressed and refolded into functionally active conformation, indicating that the tag does not interfere with the formation of internal disulfide bonds. We also describe standardized procedures for site-specific conjugation of very different payloads, such as functionalized lipids and liposomes, radionuclide chelators and radionuclides, fluorescent dyes, drug-derivatized dendrimers, scaffold proteins, biotin, and polyethyleneglycol to Cys-tagged peptides and proteins, as well as present examples of functional activity of targeted conjugates in vitro and in vivo. We expect that Cys-tag would provide new opportunities for development of targeted therapeutic and imaging agents for research and clinical use.

Abstract

Chemotactic peptides, such as Monocyte Chemotactic Protein 1 (MCP-1), play a key role in transendothelial migration of mononuclear cells during the development and progression of atherosclerotic disease. Because atherosclerotic plaques that are precursors of acute coronary events harbor abundant macrophage infiltration, we hypothesized that the detection of a high concentration of MCP-1 receptors on inflammatory cells should noninvasively identify vulnerable plaques.Atherosclerotic lesions were induced by balloon deendothelialization of the abdominal aorta, which was followed by a 0.5% cholesterol diet for 16 wk in 7 New Zealand White rabbits; 5 unmanipulated rabbits, fed normal chow for 16 wk, were used as controls. Radionuclide imaging was performed immediately after intravenous (99m)Tc-labeled MCP-1 administration and 3 h later. At the end of imaging session, aortas were explanted and submitted for estimation of quantitative MCP-1 uptake (in percentage injected dose per gram, %ID/g) and pathologic characterization.Atherosclerotic lesions were clearly visible in all hyperlipidemic animal gamma-imaging. No tracer uptake was seen in the control rabbits. The mean quantitative MCP-1 uptake in atherosclerotic lesions was 4-fold higher than that of the aortic specimens from the control rabbits (0.065 +/- 0.005 vs. 0.016 +/- 0.006; P < 0.0001). Histology confirmed a strong correlation between MCP-1 uptake and the number of macrophages in American Heart Association type II-IV lesions (r = 0.87, P < 0.0001).Noninvasive radionuclide imaging of inflammation is feasible by MCP-1 in experimentally induced atherosclerosis. It is proposed that detection of the extent of inflammation in advanced atherosclerotic plaques may allow identification of unstable plaques.

Abstract

Minocycline is an antibiotic now recognized to have antiapoptotic and antiinflammatory properties. Because of these properties, minocycline may be of benefit in reducing neuronal apoptosis from ischemia and subsequent postischemic inflammation if administered soon after a stroke. We now explore the feasibility of using (99m)Tc-annexin V, an in vivo marker of apoptosis, with SPECT to monitor the antiapoptotic effects of minocycline therapy.CB6/F1 adult male mice underwent unilateral distal middle cerebral artery occlusion (dMCA) occlusion and were imaged and sacrificed at 1, 3, 7, or 30 d after injury. Animals were given minocycline (or vehicle) 30 min and 12 h after dMCA occlusion and then given 22.5 mg/kg twice daily for up to 7 d. Before imaging, behavioral tests were performed to evaluate the neurologic function. After imaging, brains were collected for histology and assessed for the degree of apoptosis and microglial activation.(99m)Tc-Annexin V uptake in injured hemispheres was significantly decreased 2- to 3-fold by minocycline at all time points. Minocyline reduced infarct size as seen histologically and improved behavioral indices as late as 30 d. Infarct volume as seen histologically correlated with radiolabeled annexin V uptake seen by SPECT. In situ fluorescent microscopy demonstrated that annexin V bound primarily to neurons at 1 and 3 d, with a shift toward microglia by 7 and 30 d.We found that minocycline significantly reduces neuronal apoptosis and infarct size and improves neurologic outcome in mice after acute focal cortical ischemia.

Abstract

This review examines several classes of radiolabeled agents, including analogs localizing in somatostatin, benzodiazepine and dopamine receptors; analogs of progesterone and estrogen; and agents localizing in lesions with hypoxia. It concludes the status of agents advocated for detecting angiogenesis and inflammation. The current clinical status of these agents, and their potential roles in diagnosis and treatment are discussed.

Abstract

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.

Abstract

Labeled annexin V is widely used to detect cell death in vitro and in vivo. Nearly all studies have been done with annexin V derivatized via amine-directed bifunctional agents; it was thought that these molecules retained full bioactivity compared with unmodified protein. We now show that this assumption is incorrect by measuring the affinity of annexin V for cells in vitro by quantitative calcium titration under conditions of low membrane occupancy.Annexin V was modified with 4 different amine-directed agents: the N-hydroxysuccinimide esters of hydrazinonicotinic acid, mercaptoacetyltriglycine, and biotin; and with fluorescein isothiocyanate.In all cases, the membrane-binding affinity was decreased by derivatization, even at very low average stoichiometries. A statistical model based on the Poisson distribution accurately predicted the observed heterogeneity of derivatization as a function of average derivatization stoichiometry. This model also showed that multiply derivatized forms, which are the ones most likely to have compromised bioactivity, contributed disproportionately to the binding and imaging signals. The in vitro binding assay correctly predicted in vivo uptake in a mouse liver model of apoptosis for all proteins tested. The annexin V-128 protein, labeled at a single specific site at the N terminus, showed twice as much apoptosis-specific liver uptake as did all forms of annexin V derivatized randomly via amino groups.The membrane-binding activity of annexin V is much more sensitive to amine-directed chemical modification than previously realized. New annexin V molecules labeled by site-specific methods will greatly improve sensitivity for detecting cell death in vivo.

Abstract

We recently developed a cysteine-containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test (99m)Tc-HYNIC-C-tagged VEGF ((99m)Tc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment.HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. (99m)Tc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200-300 microCi, 1-2 microg protein) followed by microSPECT imaging 1 h later.Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with (99m)Tc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3+/-5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o.d. x 4; 1.14+/-0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. x 7; 1.03+/-0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of (99m)Tc/biotin-inactivated VEGF, as compared with (99m)Tc-HYNIC-VEGF.(99m)Tc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. (99m)Tc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy.

Abstract

The first aim of the study was to determine whether (99m)Tc-HYNIC-annexin V, a marker of cellular stress and apoptosis, can detect ischemic injury in patients with acute stroke. Secondly, we wished to test radiolabeled annexin's ability to monitor therapy in a rodent model of focal ischemic injury.SPECT imaging of patients was performed between 1 and 2 h after intravenous injection of 30 mCi (1,110 MBq) of tracer. Eight MFL4 (anti-FasL) antibody-treated (400 microg i.p. days 0 and 3) and 21 control adult male Sprague-Dawley rats underwent small animal SPECT imaging with 5-10 mCi (185-370 MBq) of tracer, 1 and 6 days after a 2-h intraluminal thread occlusion of the left middle cerebral artery.Two patients with acute stroke had regions of multifocal annexin uptake that correlated with sites of restricted diffusion on MRI. Anti-FasL antibody treatment significantly reduced annexin uptake by 92% with a 60% decrease in the number of caspase-8 staining (apoptotic) neurons on day 1. On day 6, treated animals had an 80% reduction in tracer uptake with a 75% decrease in infarct size as compared with controls. Annexin uptake in controls and treated animals (day 6) linearly correlated with infarct size (r (2)=0.603, p=0.0036) and the number of TUNEL-positive (apoptotic) nuclei (r (2)=0.728, p=0.00084).Annexin imaging shows foci of increased uptake at sites of ischemic injury in patients with acute stroke. Annexin imaging can assess the effects of therapy for ischemic cerebral injury in rats, suggesting its potential as a non-invasive indicator of drug efficacy in future clinical trials.

Abstract

Apoptosis is a critical factor in AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative states, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation. Improved methods to identify sites of apoptosis are increasing our understanding of the pathophysiology and treatment of these and numerous other human disorders. Here we describe the most used method for labeling annexin V, a protein with a high affinity for apoptotic cells in vitro, with technetium-99m (99mTc) as a radionuclide imaging agent that can localize and non-invasively quantify apoptosis in vivo when coupled with single-photon emission tomography. In this method, annexin V is first attached to the bifunctional chelator molecule hydrazino nicotinate (HYNIC). Once prepared, HYNIC-annexin V can be labeled with 99mTc, a widely available gamma-radiation-emitting radionuclide, for intravenous injection in as little as 30 min without the need for specialized reagents or equipment.

Abstract

(99m)Tc-Annexin V is used to image cell death in vivo via high-affinity binding to exposed phosphatidylserine. We investigated how changes in membrane-binding affinity, molecular charge, and method of labeling affected its biodistribution in normal mice and its uptake in apoptotic tissues.An endogenous Tc chelation site (Ala-Gly-Gly-Cys-Gly-His) was added to the N-terminus of annexin V to create annexin V-128. The membrane-binding affinity of annexin V-128 was then progressively reduced by 1-4 mutations in calcium-binding sites. In addition, mutations were made in other residues that altered molecular charge without altering membrane-binding affinity. All mutant proteins were labeled with (99m)Tc at the same N-terminal endogenous chelation site. Wild-type annexin V was also labeled with (99m)Tc after derivatization with hydrazinonicotinamide (HYNIC). Radiolabeled proteins were tested for biodistribution in normal mice and in mice treated to induce apoptosis of the liver.Comparison of (99m)Tc-annexin V-128 with (99m)Tc-HYNIC-annexin V showed that the protein labeled at the endogenous chelation site had the same or higher uptake in apoptotic tissues, while showing 88% lower renal uptake at 60 min after injection. The blood clearance of annexin V was unaffected by changes in either the membrane-binding affinity or the molecular charge. Kidney uptake was unaffected by changes in binding affinity. In marked contrast, uptake in normal liver and spleen decreased markedly as affinity decreased. The same pattern was observed in animals treated with cycloheximide to induce apoptosis. Control experiments with charge mutants showed that the effects seen with the affinity mutants were not due to the concomitant change in molecular charge that occurs in these mutants.(a) All four domains of annexin V are required for optimal uptake in apoptotic tissues; molecules with only 1 or 2 active domains are unlikely to be suitable for imaging of cell death in vivo. (b) Uptake in normal liver and spleen is specific (dependent on phosphatidylserine-binding affinity), whereas renal uptake is nonspecific. (c) (99m)Tc-Annexin V-128 detects cell death as well as (99m)Tc-HYNIC-annexin V, while showing 88% less renal retention of radioactivity due to much more rapid urinary excretion of radioactivity.

Abstract

Cancer chemotherapy enhances the apoptosis, whereas apoptosis is a suicidal mechanism requiring energy. We determined the relationship between apoptosis and glucose utilization during cancer chemotherapy using (99m)Tc-annexin V ((99m)Tc-annexin A5) and (18)F-FDG and compared their uptake with histologic findings in a rat tumor model.Allogenic hepatoma cells (KDH-8) were inoculated into the left calf muscle of male Wistar rats (WKA). Eleven days after the inoculation, the rats were randomly divided into 3 groups: The first group (n = 7) received a single dose of gemcitabine (90 mg/kg, intravenously), the second group (n = 8) received cyclophosphamide (150 mg/kg, intraperitoneally), and the third group (n = 7) was untreated and served as the control group. We injected (99m)Tc-annexin V 48 h after the chemotherapy and then injected (18)F-FDG to all rats 1 h before sacrifice. Six hours after (99m)Tc-annexin V injection, the rats were sacrificed and the organs, including the tumor, were removed and radioactivity was counted. The radioactivities of (18)F and (99m)Tc in the organs were determined using normalization by tissue weight. Histologic evaluation by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method and the immunostaining of glucose transporter-1 (GLUT-1) were also performed to obtain the indices of apoptosis and glucose utilization, respectively. The rate of positively stained cells was calculated and analyzed statistically.After chemotherapy using gemcitabine and cyclophosphamide, the (99m)Tc-annexin V uptake (percentage injected dose per gram x kg [(%ID/g) x kg]; mean +/- SD) in tumor increased significantly (0.062 +/- 0.012 (%ID/g) x kg in the gemcitabine-treated group and 0.050 +/- 0.012 (%ID/g) x kg in the cyclophosphamide group vs. 0.031 +/- 0.005 (%ID/g) x kg in the control group; P < 0.01). In contrast, the (18)F-FDG in tumor decreased significantly (0.483 +/- 0.118 (%ID/g) x kg in the gemcitabine group and 0.583 +/- 0.142 (%ID/g) x kg in the cyclophosphamide group) compared with that in the control group (0.743 +/- 0.084 (%ID/g) x kg; P < 0.01). In addition, (18)F-FDG uptake in tumor negatively correlated with (99m)Tc-annexin V uptake (r = -0.75; P < 0.01). In the gemcitabine and cyclophosphamide groups, the rate of TUNEL positively stained cells was significantly higher than that in the control group (10.2% +/- 1.7% and 8.0% +/- 1.5% vs. 5.2% +/- 1.5%; P < 0.01), whereas the GLUT-1 expression level showed no definite changes in histologic analyses.These data indicate that an enhanced apoptotic reaction correlated with suppressed tumor glucose utilization after cytotoxic chemotherapy as determined using radiotracers and histologic evaluation. The increase in (99m)Tc-annexin V and the decrease in (18)F-FDG in tumor can be useful markers for predicting therapeutic outcomes and for prognosis at the early stage of chemotherapy.

Abstract

CD4+CD25+ regulatory T (Treg) cells are potent modulators of alloimmune responses. In murine models of allogeneic bone marrow transplantation, adoptive transfer of donor CD4+CD25+ Treg cells protects recipient mice from lethal acute graft-versus-host disease (aGVHD) induced by donor CD4+CD25- T cells. Here we examined the differential effect of CD62L+ and CD62L- subsets of CD4+CD25+ Treg cells on aGVHD-related mortality. Both subpopulations showed the characteristic features of CD4+CD25+ Treg cells in vitro and did not induce aGVHD in vivo. However, in cotransfer with donor CD4+CD25- T cells, only the CD62L+ subset of CD4+CD25+ Treg cells prevented severe tissue damage to the colon and protected recipients from lethal aGVHD. Early after transplantation, a higher number of donor-type Treg cells accumulated in host mesenteric lymph node (LN) and spleen when CD4+CD25+CD62L+ Treg cells were transferred compared with the CD62L- subset. Subsequently, CD4+CD25+CD62L+ Treg cells showed a significantly higher capacity than their CD62L- counterpart to inhibit the expansion of donor CD4+CD25- T cells. The ability of Treg cells to efficiently enter the priming sites of pathogenic allo-reactive T cells appears to be a prerequisite for their protective function in aGVHD.

Abstract

In tumors the process of apoptosis occurs over an interval of time after chemotherapy. To determine the best timing for detecting apoptosis in vivo with (99m)Tc-annexin V after chemotherapy, we examined the changes in (99m)Tc-annexin V accumulation over time in comparison with those of caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) expression level after cyclophosphamide treatment in an experimental model.Hydrazinonicotinamide (HYNIC)-annexin V was labeled with (99m)Tc ((99m)Tc-annexin V). Rats were inoculated with allogenic hepatoma cells (KDH-8) into the left calf muscle. Eleven days after the inoculation, the rats were randomly divided into the group receiving a single dose of cyclophosphamide (150 mg/kg intraperitoneally) and the control group. (99m)Tc-Annexin V (18.5 MBq [0.5 mCi] per rat) was injected intravenously in the rats 4, 12, and 20 h after the treatment and also to the control rats (n = 5 in each group). Radioactivity in tissues was determined 6 h after (99m)Tc-annexin V injection. Immunostaining of caspase-3 and TUNEL were performed to detect apoptosis, and the rates of positively stained cells were calculated.(99m)Tc-Annexin V accumulation in tumors significantly increased at 20 h (0.077 +/- 0.007 [%ID/g] x kg, where %ID/g = percentage injected dose per gram) but not at 4 or 12 h (0.048 +/- 0.008 and 0.052 +/- 0.014 [%ID/g] x kg, respectively) after cyclophosphamide treatment. (99m)Tc-Annexin V accumulation in tumors and the rate of apoptotic cells determined by caspase-3 immunostaining and TUNEL were significantly higher in treated rats 20 h after cyclophosphamide treatment as compared with control rats.The effective detection of apoptotic tumor response with (99m)Tc-annexin V required 20 h after cyclophosphamide treatment in an experimental model. The present results provide an important basis for determining the best timing of annexin V imaging after the start of chemotherapy in a clinical setting.

Abstract

Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively.HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(121) (Hu-VEGF [vascular endothelial growth factor]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice.There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively).(99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.

Molecular imgaging with single photon emission computed tomography - How new tracers can be employed in the nuclear medicine clinicIEEE ENGINEERING IN MEDICINE AND BIOLOGY MAGAZINEBlankenberg, F. G.2004; 23 (4): 51-57

Abstract

Craniosynostosis, the premature fusion of cranial sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-frontal suture fusion and sagittal suture patency to globally examine apoptosis in cranial sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-frontal suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-frontal and sagittal suture complexes during the period of predicted posterior-frontal suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-frontal and sagittal sutures during the period of predicted posterior-frontal suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-frontal suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing sutures is similar, the pathways mediating apoptosis within each suture are distinct.

Abstract

In this study we wished to determine whether technetium-99m annexin V, an in vivo marker of cellular injury and death, could be used to noninvasively monitor neuronal injury following focal middle cerebral artery (MCA) occlusion/reperfusion injury. Sixteen adult male Sprague-Dawley rats (along with four controls) underwent left (unilateral) MCA intraluminal beaded thread occlusion for 2 h followed by reperfusion. One hour following tail vein injection of 5-10 mCi of (99m)Tc-annexin V, animals underwent either single-photon emission computerized tomography (SPECT) or autoradiography followed by immunohistochemical analyses. There was abnormal, bilateral, multifocal uptake of (99m)Tc-annexin V in each cerebral hemisphere as seen by both SPECT and autoradiography at 4 h and 1, 3, and 7 days after initiation of occlusion. The average maximal annexin V uptake at 4 h was 310%+/-85% and 365%+/-151% above control values (P<0.006) within the right and left hemispheres, respectively, peaking on day 3 with values of 925%+/-734% and 1,194%+/-643% (P<0.03) that decreased by day 7 to 489%+/-233% and 785%+/-225% (P<0.01). Total lesional volume of the left hemisphere was 226%, 261%, and 451% ( P<0.03) larger than the right at 4, 24, and 72 h after injury, respectively. Annexin V localized to the cytoplasm of injured neurons ipsilateral to the site of injury as well as to otherwise normal-appearing neurons of the contralateral hemisphere as confirmed by dual fluorescent microscopy. It is concluded that there is abnormal bilateral, multifocal annexin V uptake, greater on the left than on the right side, within 4 h of unilateral left MCA ischemic injury and that the uptake peaks at 3 days and decreases by 7 days after injury. This pattern suggests that neuronal stress may play a role in the response of the brain to focal injury and be responsible for annexin V uptake outside the region of ischemic insult.

Abstract

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.

Abstract

A deficiency or an excess of programmed cell death (apoptosis) is an integral component of autoimmune disorders, organ and bone marrow transplant rejection, and cancer. A technique to image programmed cell death would be useful in the development of drugs to treat these and others diseases, and to monitor the effectiveness of therapy. The most widely studied agent for the in vivo study of apoptosis is radiolabeled annexin V, an endogenous protein labeled with technectium-99m, now undergoing clinical trials in both Europe and the United States. While annexin V has been studied extensively in humans the precise mechanism(s) of uptake of this agent in vivo is unclear and needs further study. Other agents are also underdevelopment including radiolabeled forms of Z-VAD.fmk, a potent inhibitor of the enzymatic cascade intimately associated with apoptosis. MR imaging techniques and tracers also hold promise as methods to monitor apoptotic cell death. In this article we will review these and other imaging technologies for the non-invasive imaging of cell death. The mechanism(s) and latest data on the conditions in which cellular stress and early apoptosis occur will also be discussed in detail including potential new strategies for the targeting and novel therapeutic interventions of tissues and organs undergoing stress or apoptosis when cell salvage is still possible.

Abstract

A technique to image programmed cell death would be useful both in clinical care and in drug development. The most widely studied agent for the in vivo study of apoptosis is radiolabeled annexin V, an endogenous protein labeled with technectium-99m, now undergoing clinical trials in both Europe and the United States. While annexin V has been studied extensively in humans the precise mechanism(s) of uptake this agent in vivo is unclear and needs further study. Other agents are also under development, including radiolabeled forms of Z-VAD.fmk, a potent inhibitor of the enzymatic cascade intimately associated with apoptosis. In addition other technologies, such as diffusion weighted magnetic resonance imaging and magnetic resonance imaging with contrast agents, such as small paramagnetic iron oxide particles coated with peptides have also been advocated as methods to monitor apoptotic cell death. The potential applications of imaging apoptosis as a marker of early response to therapy in cancer, acute cerebral and myocardial ischemic injury and infarction, immune mediated inflammatory disease and transplant rejection are reviewed.

Abstract

Molecular Imaging technologies will have a profound impact on both basic research and clinical imaging in the near future. As the field covers many different specialties and scientific disciplines it is not possible to review all in a single article. In the current article we will turn our attention to those modalities that are either currently in use or in development for the medical imaging clinic.

Abstract

With the advent of therapies aimed at young patients with cystic fibrosis, who have mildly reduced pulmonary function, the need for improved outcome measures that discriminate treatment effects has become important. Pulmonary function measurements or chest high-resolution computed tomography (HRCT) scores have been separately used to assess interventions. We evaluated these modalities separately and together during a treatment study to develop a more sensitive outcome measure. In a 1-year trial, 25 children randomized either to daily Pulmozyme or to normal saline aerosol were evaluated at randomization and at 3 and 12 months. Outcome variables were pulmonary function test (PFT) results, a global HRCT score, and a composite score incorporating PFTs and HRCT scoring. Regression analyses with generalized estimating equations permitted estimation of the difference in treatment effect between groups over time for each outcome. The largest difference in treatment effects observed at 12 months, measured by the percentage change from baseline, were with the composite total and maximal CT/PFT scores (35.4 and 30.4%), compared with mean forced expiratory flow during the middle half of the FVC (FEF25-75%) (13.0%) and total and maximal global HRCT scores (6.2%, 7.2%). The composite total and maximal CT/PFT scores were the most sensitive outcome measures for discriminating a treatment effect in children with cystic fibrosis with normal or mildly reduced pulmonary function during a 1-year trial of Pulmozyme.

Abstract

To automatically derive the degree of air trapping in mild cystic fibrosis (CF) disease from high-resolution CT (HRCT) data, and to evaluate the discriminating power of the measurement.The data consist of six pairs of anatomically matched tomographic slices, obtained during breath-holding in triggered HRCT acquisitions. The pairs consist of an inspiratory slice, at > or = 95% of slow vital capacity, and an expiratory slice at near residual volume (nRV). The subjects are 25 patients with mild CF and 10 age-matched, normal control subjects.Lung segmentation is automatic. The limits defining air trapping in the expiratory slices are determined by the distribution of densities in the expanded lung. They are modulated by density changes between expiration and inspiration. Air trapping defects consist of contiguous low-density voxels. The difference between patients and control subjects was evaluated in comparison to pulmonary function test (PFT) results and lung density distribution descriptors (global density descriptors).In mild CF, air trapping does not correlate with global PFT results, except for the ratio of residual volume (RV) to total lung capacity (TLC); however, the size of air trapping defects was the best discriminator between patients and control subjects (p < 0.005). Of PFT results, only RV/TLC reached significance at p < 0.05. The global density descriptors reached near significance in the nRV images only.Air trapping defined as defect size and measured in an objective automated manner is a powerful discriminator for mild CF.

Abstract

Inflammation and cell death are two important components of myocarditis. We evaluated the distribution of inflammation and apoptotic cell death in rats with autoimmune myocarditis using two radiotracers - technetium-99m Hynic-annexin V ((99m)Tc-annexin) as a marker of apoptotic cell death and carbon-14 deoxyglucose ((14)C-DG) as a marker of inflammation - in comparison with histologic findings. Three, 7 and 14 weeks after immunization with porcine cardiac myosin (acute, subacute, and chronic phases, respectively) (99m)Tc-annexin and (14)C-DG were injected. The uptake in the total heart was determined as the percentage of injected dose per gram (% ID/g) by tissue counting. Dual-tracer autoradiography with (99m)Tc-annexin and (14)C-DG was performed. The distribution of each of these agents was compared with the results of hematoxylin and eosin staining to identify areas of inflammation, and TUNEL staining to identify areas of apoptosis. Total cardiac uptake of (99m)Tc-annexin in the acute phase of myocarditis was significantly higher than that in normal rats (1.28%+/-0.30% vs 0.46%+/-0.01%; P<0.0001); it then decreased in the subacute phase and reached normal levels (0.56%+/-0.08% vs 0.60%+/-0.08%; P=NS). Total cardiac uptake of (14)C-DG in the acute phase of myocarditis was significantly higher than that in normal rats (2.78%+/-0.95% vs 1.02%+/-0.25%; P<0.0001); it then decreased in the subacute phase, but still remained higher than in controls (2.06%+/-0.52% vs 1.37%+/-0.46%; P<0.05). Using autoradiography and staining of tissue specimens, it was found that most histologic inflammatory foci corresponded to areas of high (14)C-DG uptake; some also corresponded to areas of high (99m)Tc-annexin uptake in the acute phase of myocarditis. (99m)Tc-annexin localization was strongly correlated with the number of TUNEL-positive cells (P<0.0001, r=0.83), but the uptake of (14)C-DG showed no relationship with it. There is a marked difference in the distribution of inflammation and apoptotic cell death in the myocardium of animals with immune myocarditis. These changes are mirrored by the localization of (14)C-DG and (99m)Tc-annexin. Sites of inflammation and zones of apoptotic cell death change over the course of immune myocarditis.

Abstract

Annexin V, a human protein with a high affinity for phosphatidylserine, has been labeled with (99m)Tc to detect apoptosis in vivo. To determine the effectiveness of imaging with this agent as a reflection of the degree of apoptosis after the first dose of chemotherapy, we studied rats with an engrafted hepatoma.Annexin V was labeled with (99m)Tc (specific activity, 3.0 MBq/ micro g protein). Eleven days after being inoculated with allogenic hepatoma cells (KDH-8) in the left calf muscle, the rats were randomized to receive a single dose of cyclophosphamide (150 mg/kg intraperitoneally) or to serve as controls. (99m)Tc-annexin V was injected 20 h later. Radioactivity in tissues was determined 6 h after injection of (99m)Tc-annexin V. Tumor uptake of (14)C-iodoanitpyrine was determined as a marker of tumor blood flow. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) of tissue harvested at necropsy was performed to detect apoptosis in the tumor.Cyclophosphamide treatment significantly increased the tumor uptake (percentage activity of injected dose per gram of tissue after normalization to the animal's weight [%ID/g/kg]) of (99m)Tc-annexin V (0.070 +/- 0.007 %ID/g/kg for treated rats and 0.046 +/- 0.009 %ID/g/kg for controls, P < 0.001). (14)C-iodoantipyrine uptake was similar in the treated and untreated groups. The number of TUNEL-positive cells in the tumor was significantly larger in the treated rats (297.70 +/- 50.34 cells/mm(2)) than in the control rats (168.45 +/- 23.60 cells/mm(2), P < 0.001). Tumor uptake of (99m)Tc-annexin V correlated with the number of TUNEL-positive cells in the tumor (r = 0.712; P < 0.001).Tumor uptake of (99m)Tc-annexin V was significantly increased by a single dose of cyclophosphamide treatment, and the increase was concordant with the number of TUNEL-positive cells in the tumor. The current results are suggestive of the utility of (99m)Tc-annexin V as a noninvasive means to assess tumor response, although further testing, including clinical evaluation, is required.

Abstract

Rheumatoid arthritis is associated with chronic synovial inflammation due to the abnormal accumulation of macrophages and autoreactive T lymphocytes in joints. The autoreactive cells cause an inflammatory proapoptotic response to self-antigens resulting in eventual bone, cartilage, and soft-tissue loss and destruction. The goal of our study was to determine the timing and intensity of apoptosis in joints using 99mTc-labeled annexin V, an in vivo marker of apoptosis, in a murine model of immune arthritis.We used 99mTc-annexin V and autoradiography to study the extent and severity of apoptosis in the front and rear paws of DBA/1 mice with type II collagen-induced rheumatoid arthritis.Compared with control values (n = 10), there was a significant (P < 0.002) nearly 3-fold increase in uptake of 99mTc-annexin V in the front foot pads, rear toes, rear foot pads, and heels at the time of maximal extremity swelling as determined by serial caliper measurements at 4 wk after inoculation with type II bovine collagen (n = 9). The front toes had a 5- to 6-fold increase in uptake compared with control values (P < 0.001). Histologic analysis revealed only scattered rare lymphocytes in the periarticular soft tissues, without joint destruction. Dual autoradiography with 125I-bovine serum albumin as a control showed that 99mTc-annexin V localization was specific. Treatment with methylprednisolone for 1 wk (n = 8) at 4 wk after immunization with type II collagen decreased 99mTc-annexin V uptake by 3- to 6-fold compared with control values (P < 0.002).99mTc-annexin V can detect collagen-induced immune arthritis and its response to steroid therapy before joint destruction.

Abstract

With the emergence of the new field of molecular imaging, there is an increasing demand for development of sensitive and safe novel imaging agents that can be rapidly translated from small animal models into patients. Nuclear medicine and positron emission tomography (PET) techniques have the ability to detect and serially monitor a variety of biologic and pathophysiologic processes, usually with tracer quantities of radiolabeled peptides, drugs, and other molecules at doses free of pharmacologic side effects, unlike the current generation of intravenous agents required for magnetic resonance (MR) and computed tomography (CT) scanning. In this article, we will review a representative sampling of the wide array of radiopharmaceuticals developed specifically for nuclear medicine radionuclide imaging that have been approved for clinical use, and those in pre-clinical trials. We will also review the existing strategies used to select the appropriate biologic markers and targets for radionuclide labeling that have been employed in the development of novel radiotracers and the imaging of small animals with new microSPECT (single photon emission computed tomography) technologies.

Abstract

Monocyte chemoattractant protein-1 (MCP-1) is associated with the development of graft coronary artery disease (GCAD) following cardiac transplantation. This study assessed whether technetium 99m ((99m)Tc)-labeled MCP-1 binds its receptors in chronic cardiac transplants and thereby provides a potential modality to assess GCAD.Allogeneic (PVG-->ACI, n = 9) and syngeneic (ACI-->ACI, n = 9) rat heterotopic heart transplants were performed. Allograft recipients were treated with 7.5 mg/kg per day of Cyclosporin A for 10 days until tolerance was achieved. After 90 days, animals were injected intravenously with (99m)Tc-MCP-1 and killed after 1 hour. Radioactivity of heart tissues was measured and standardized to uptake in the overall blood pool. Two-dimensional (99m)Tc-MCP-1 uptake (autoradiographs) was imaged by exposing 50-microm sections on a phosphoimager overnight. ED-1 staining of monocyte/macrophages was performed on serial sections. Additional sections were stained with elastin von Gieson and hematoxylin. Hearts were scored for luminal narrowing and intima/media ratio (I/M) with computerized image analysis.Allografts exhibited significantly more luminal narrowing (22.5 +/- 10.7% vs 2.6 +/- 4.6, p = 0.0005) and higher I/M (0.173 +/- 0.151 vs 0.015 +/- 0.029, p = 0.0088) than isografts. The ratio of (99m)Tc-MCP-1 uptake in allografts (1.04 +/- 0.4) was greater than that of isograft controls (0.72 +/- 0.11, p = 0.03). Pixel counts of autoradiographs and ED-1-stained sections demonstrated a modest correlation between the two (R(2) = 0.50). No significant differences were seen in acute rejection scores.(99m)Tc-MCP-1 uptake was higher in allografts vs isografts and was consistent with a greater degree of GCAD. These data demonstrating increased radiopharmaceutical uptake in hearts with GCAD provide a foundation for the development of a potentially non-invasive imaging assay of this disease process in heart transplantation.

Abstract

The revolution in molecular imaging techniques is profoundly changing the understanding of the pathophysiology and treatment of atherosclerosis. With these rapid changes there is an increasing demand for development of sensitive and well tolerated novel imaging agents that can be rapidly translated from small animal models into patients with atherosclerosis. Nuclear medicine and positron emission tomography techniques have the ability to detect and serially monitor a variety of biologic and pathophysiologic processes usually with tracer quantities of radiolabeled peptides, drugs, and other molecules at dosages free of pharmacologic adverse effects unlike the current generation of intravenous agents required for magnetic resonance imaging (MRI) and computed axial tomography (CT) scanning. A representative sampling of the wide array of radiopharmaceuticals developed specifically for radionuclide imaging of atherosclerosis, that have been approved for clinical use and those in pre-clinical trials, have been reviewed in this article. The presence of an inflammatory stimulus increases expression of CC (cysteine-cysteine motif) chemokine receptor (CCR)-2 on monocytes and macrophages, and somatostatin receptors on T lymphocytes. Radiolabeled monocyte chemoattractant protein (MCP)-1 binds with high affinity to CCR-2 and can be used to detect subacute and chronic inflammatory lesions. Similarly, radiolabeled octreotide or depreotide can be used to detect activated T lymphocytes which may identify the vulnerable plaque. Animal models indicate that (99m)Tc-annexin V, (125)I-MCP-1 and [(18)F]-fluoro-2-deoxyglucose are effective in identifying apoptotic cell death, macrophage infiltration and metabolic activity in atheromatous lesions, respectively. Expression of alpha(v)beta(3) integrin is increased in activated endothelial cells and vascular smooth muscle cells after vascular injury, and alpha(v)beta(3) integrin is minimally expressed on smooth muscle cells and is not expressed on quiescent epithelial cells. Radiolabeled high-affinity peptides can be used to target the alpha(v)beta(3) integrin and visualize areas of vascular damage. Advances in technology such as the micro-single photon emission computed tomography (microSPECT) have the potential to overcome the drawbacks of older CT and MRI methodologies, such as lack of biologically relevant ligands and compatible blood pool contrast agents for imaging. Despite these advances in imaging technology, the small size of atheromatous lesions makes it difficult to detect using external imaging techniques. Therefore, recently there has been renewed interest in the use of intravascular catheter-based radiation detectors.

Abstract

Migration of monocytes into the arterial wall is an early finding of atherosclerosis. Monocytes are attracted to sites of vascular endothelial cell injury, the initiating event in the development of atheromatous disease, by a chemokine known as monocyte chemoattractant protein-1 (MCP-1). Injured vascular endothelial and smooth muscle cells selectively secrete MCP-1.This study was performed to determine if radiolabeled MCP-1 would co-localize at sites of monocyte/macrophage concentration in an experimental model of transplant-induced vasculopathy in diabetic animals.Hearts from 3-month-old male Zucker rats, heterozygote (Lean) or homozygote (Fat) for the diabetes-associated gene fa, were transplanted into the abdomens of genetically matched recipients. Lean and Fat animals were then fed normal or high-fat diets for 90 days.At 90 days significant increases (P < 0.013) of MCP-1 graft uptake were seen at imaging and confirmed on scintillation gamma well counting studies in Lean (n = 5) and Fat (n = 12) animals, regardless of diet, 400 % and 40 %, above control values, respectively. MCP-1 uptake of native and grafted hearts correlated with increased numbers of perivascular macrophages (P < 0.02), as seen by immunostaining with an antibody specific for macrophages (ED 2).Radiolabeled MCP-1 can detect abnormally increased numbers of perivascular mononuclear cells in native and grafted hearts in prediabetic rats. MCP-1 may be useful in the screening of diabetic children for early atherosclerotic disease.

Abstract

Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.

Abstract

Annexin V binds phosphatidylserine moieties on apoptotic cells. This study reports the initial experience at Stanford University Medical Center with 99mTc-labeled annexin V imaging as a noninvasive measure of apoptosis in acute cardiac rejection. Ten cardiac transplant patients had 99mTc Annexin V imaging and endomyocardial biopsy (EMB) performed within 24 h. No complications related to 99mTc annexin V administration occurred. Eight patients had ISHLT grade of acute rejection of 1A or less. Five patients had two or more areas of uptake noted in the right ventricle on imaging studies. Two of these patients had positive biopsies: one patient had grade 2 rejection with two focal uptake areas and another had grade 3A rejection with three foci. An additional five patients had either one or zero hot spot areas and corresponding negative EMBs. 99mTc-annexin V appears to be well tolerated and may identify patients with acute cardiac rejection.

Abstract

Monocytes/macrophages (Mphis), the predominant cell types in subacute and chronic inflammation, are attracted to and activated by monocyte chemotactic peptide-1 (MCP-1). Mphis promote the resolution of inflammation through the induction of apoptosis and phagocytosis of senescent (spent) and bystander (superfluous) granulocytes. We wished to determine whether MCP-1, which selectively binds to Mphis, could be used to image subacute and chronic inflammation. We also sought to image granulocyte apoptosis within these lesions with technetium-99m labeled annexin V, a marker of apoptotic cells. Sterile inflammation was induced in 45 12-week-old male Sprague-Dawley rats by deep intramuscular injection of turpentine into the right thigh. Groups of four to six animals were then imaged 1 h after tail vein injection of 37-148 MBq (1-4 mCi) of 99mTc-labeled MCP-1 or annexin V 1-14 days after turpentine treatment. Image analysis showed significantly greater activity of both MCP-1 and annexin V in inflamed thighs than in control thighs (165%-290% and 188%-313%, respectively; P<0.01) on days 1-5 after turpentine injection. Dual autoradiography in animals co-injected with iodine-125 labeled bovine serum albumin on days 1 and 4 showed specific location of MCP-1 to infiltrating Mphis while annexin V localized to focal zones of apoptosis within granulocytic infiltrates adjacent to abscess cavities. Scintillation well counting on day 5 demonstrated significantly higher (P<0.005) ratios of abscess to control thigh specific activities for MCP-1 (5.83+/-2.17) and annexin V (9.24 +/- 2.8) as compared to 125I-labeled bovine serum albumin (3.11 +/- 0.65). No significant increases in uptake were noted at imaging or ex vivo analyses on days 13 and 14, when lesions were predominately fibrotic. It is concluded that 99mTc-labeled MCP-1 and 99mTc-labeled annexin V both localize in zones of subacute inflammation, reflecting the density of Mphis and the incidence of apoptotic granulocytes, respectively. These agents may be useful in the characterization of subacute inflammation.

Abstract

The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells.Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant.Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts.These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.

Abstract

To evaluate a high-resolution computed tomography (HRCT) scoring system, clinical parameters, and pulmonary function measurements in patients with cystic fibrosis (CF) before and after therapy for a pulmonary exacerbation.Patients (n = 17) were evaluated by spirometer-triggered HRCT imaging, clinical parameters, and pulmonary function tests (PFTs) before and after treatment. HRCT scans were reviewed by 3 radiologists using a modified Bhalla scoring system.Bronchiectasis, bronchial wall thickening, and air trapping were identified in all subjects on initial evaluation. The initial total HRCT score correlated significantly with the Brasfield score (r = -.91, P

Abstract

Intramedullary apoptosis of hematopoietic tissue is believed to play a major role in the pathophysiology of myelodysplastic syndrome. Annexin V, a specific marker of the early to intermediate phases of apoptosis, has been applied to the in vitro study of bone marrow aspirates. A noninvasive measure of intramedullary apoptosis in vivo that could serially monitor the clinical progression of myelodysplastic syndrome may be helpful.We used 99mTc-radiolabeled annexin V and radionuclide gamma camera imaging to serially study the sites, extent, and severity of intramedullary apoptosis induced by cyclophosphamide treatment.Intravenously administered radiolabeled annexin V localized preferentially in the femur, pelvis, vertebrae, and spleen; increased uptake in these organs was easily visualized as early as 8 h after injection of 100 mg/kg cyclophosphamide in 8- to 10-wk-old animals. Higher doses of cyclophosphamide (150 mg/kg) in animals of the same age increased annexin V uptake in the bone marrow and splenic tissue and delayed recovery of these organs as seen histologically compared with lower doses. Older animals, 5-6 mo old, showed a slower response to cyclophosphamide treatment and delayed recovery of bone marrow and splenic tissues.Radiolabeled annexin V can be used to detect and directly quantify the degree of intramedullary and splenic apoptosis in a noninvasive fashion using current clinical radionuclide imaging equipment. Annexin V imaging may be useful clinically in the diagnosis and management of myelodysplastic syndrome.

Will imaging of apoptosis play a role in clinical care? A tale of mice and menAPOPTOSISBlankenberg, F. G., Strauss, H. W.2001; 6 (1-2): 117-123

Abstract

Programmed cell death (apoptosis) plays a role in the pathophysiology of many diseases and in the outcome of treatment. Apoptosis is the likely mechanism behind the cytoreductive effects of standard chemotherapeutic and radiation treatments, rejection of organ transplants, cellular damage in collagen vascular disorders, and delayed cell death due to hypoxic-ischemic injury in myocardial infarction and neonatal hypoxic ischemic injury. Observations about the role of apoptosis have fueled the development of novel agents and treatment strategies specifically aimed at inducing or inhibiting apoptosis. Despite these research developments there are no clinical entities where specific measures of apoptosis are used in either diagnosis or patient management. Part of the difficulty in bridging the gap between the basic science understanding of apoptosis and the clinical application of this information is the lack of a sensitive marker to monitor programmed cell death in association with disease progression or regression. Technetium-99m labeled annexin V localizes at sites of apoptosis in-vivo, due to its nanomolar affinity for membrane bound phosphatidylserine. Radiolabeled annexin V imaging permits identification of the site and extent of apoptosis in experimental animals. Annexin V has been successfully used in animal models to image organ transplant rejection, characterize successful therapy of tumors, pinpoint acute myocardial infarction, and identify hypoxic ischemic brain injury of the newborn and adult. Early studies in human subjects suggest that 99mTc annexin imaging will be also be useful to identify rejection in transplant recipients, localize acute myocardial infarction, and characterize the effectiveness of a single treatment in patients with tumors. This review describes the imaging approaches to detect and monitor apoptosis in-vivo that are presently in early clinical trials. The preliminary data are extrapolated to identify conditions where apoptosis imaging may be valuable in clinical decision making. These conditions include: transplant rejection; hypoxic/ischemic injury of heart and brain; and determining the efficacy of therapy in cancer, heart failure and osteoporosis.

Abstract

Apoptosis consists of a complex set of biochemical events initiated by an array of different stimuli and enzymatic pathways. There is a set of common morphologic and biochemical features of apoptosis that could be exploited as hot or cold targets to image cardiovascular apoptosis. First, the authors review the potential array of targets that can be used to identify apoptosis. Then, the authors examine the history and current status of radiolabeled annexin V, the agent currently used to image apoptosis.

Abstract

Apoptosis is thought to occur during immune-mediated acute rejection of cardiac allografts. In vitro studies have shown that zinc inhibits the activity of the proapoptotic enzyme caspase-3. We hypothesized that ZnCl(2) would reduce acute cardiac rejection in vivo via the blockade of caspase-3-dependent apoptosis. (99m)Tc-labeled annexin V was used to measure apoptosis in cardiac allografts through nuclear imaging. Annexin V binds to phosphatidylserines, which are externalized to the outer membrane of apoptotic cells.Twenty-seven PVG rat hearts were transplanted heterotopically into the abdomen of untreated ACI rats as controls (group 1). Fifteen were scanned and euthanized on postoperative day 4, and 12 were assessed for graft survival. Group 2 and 3 rats (n=15 each) received 1 and 5 mg/kg ZnCl(2) BID IP, respectively. Nine of each of these groups were scanned and euthanized on postoperative day 4, and 6 were studied for allograft survival. Group 4 rats (n=3) received isografts. Region-of-interest analysis demonstrated a dose-dependent reduction in (99m)Tc annexin uptake in ZnCl(2)-treated allografts: 2.43+/-0.37% for group 1, 1. 97+/-0.41% for group 2, 1.21+/-0.47% for group 3, and 0.55+/-0.19% for group 4 (ANOVA, P:=0.001). Graft survival times of 6.4+/-1.7, 9. 3+/-3.0, and 11.5+/-3.4 days for groups 1, 2, and 3, respectively, were also observed (ANOVA, P:=0.001). Caspase-3 activity in the allografts showed a 3.7-fold reduction in group 3 animals compared with group 1 animals (P:=0.004).Apoptosis that occurs in acute cardiac allograft rejection is reduced with ZnCl(2) in a dose-dependent manner via caspase-3 inhibition.

Abstract

Delayed cell loss in neonates after cerebral hypoxic-ischemic injury (HII) is believed to be a major cause of cerebral palsy. In this study, we used radiolabeled annexin V, a marker of delayed cell loss (apoptosis), to image neonatal rabbits suffering from HII.Twenty-two neonatal New Zealand White rabbits had ligation of the right common carotid artery with reduction of inspired oxygen concentration to induce HII. Experimental animals (n=17) were exposed to hypoxia until an ipsilateral hemispheric decrease in the average diffusion coefficient occurred. After reversal of hypoxia and normalization of average diffusion coefficient values, experimental animals were injected with (99m)Tc annexin V. Radionuclide images were recorded 2 hours later.Experimental animals showed no MR evidence of blood-brain barrier breakdown or perfusion abnormalities after hypoxia. Annexin images demonstrated multifocal brain uptake in both hemispheres of experimental but not control animals. Histology of the brains from experimental animals demonstrated scattered pyknotic cortical and hippocampal neurons with cytoplasmic vacuolization of glial cells without evidence of apoptotic nuclei by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. Double staining with markers of cell type and exogenous annexin V revealed that annexin V was localized in the cytoplasm of scattered neurons and astrocytes in experimental and, less commonly, control brains in the presence of an intact blood-brain barrier.Apoptosis may develop after HII even in brains that appear normal on diffusion-weighted and perfusion MR. These data suggest a role of radiolabeled annexin V screening of neonates at risk for the development of cerebral palsy.

Abstract

[(99m)Tc]Annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. To simplify the preparation and labeling of annexin V for nuclear-medicine studies, we have investigated the addition of peptide sequences that will directly form endogenous chelation sites for (99m)Tc. Three mutant molecules of annexin V, called annexin V-116, -117, and -118, were constructed with N-terminal extensions of seven amino acids containing either one or two cysteine residues. These molecules were expressed cytoplasmically in Escherichia coli and purified to homogeneity with a final yield of 10 mg of protein/L of culture. Analysis in a competitive binding assay showed that all three proteins retained full binding affinity for erythrocyte membranes with exposed phosphatidylserine. Using SnCl(2) as reducing agent and glucoheptonate as exchange agent, all three proteins could be labeled with (99m)Tc to specific activities of at least 50-100 microCi/microg. The proteins retained membrane binding activity after the radiolabeling procedure, and quantitative analysis indicated a dissociation constant (K(d)) of 7 nmol/L for the annexin V-117 mutant. The labeling reaction was rapid, reaching a maximum after 40 min at room temperature. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro. Proteins labeled to a specific activity of 25-100 microCi/microg were injected intravenously in mice at a dose of 100 microg/kg, and biodistribution of radioactivity was determined at 60 min after injection. Uptake of radioactivity was highest in kidney and liver, consistent with previous results obtained with wild-type annexin V. Cyclophosphamide-induced apoptosis in vivo could be imaged with [(99m)Tc]annexin V-117. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc without altering its high affinity for cell membranes. These annexin V derivatives may be useful for in vivo imaging of cell death.

Abstract

Apoptosis is a genetically controlled, energy-dependent process which removes unwanted cells from the body. Because of its orderly progression, apoptosis is also known as programmed cell death or cell suicide. Once initiated, apoptosis is characterized by a series of biochemical and morphological changes involving the cytoplasm, nucleus and cell membrane. Cytoplasmic changes include cytoskeletal disruption, cytoplasmic shrinkage and condensation; prominent changes in the nucleus include peripheral chromatin clumping and inter-nucleosomal DNA cleavage (DNA ladder formation); and membrane changes include the expression of phosphatidylserine on the outer surface of the cell membrane and blebbing (resulting in the formation of cell membrane-bound vesicles or apoptotic bodies). These events allow the cell to digest and package itself into membrane-bound packets containing autodigested cytoplasm and DNA, which can then be easily absorbed by adjacent cells or phagocytes. An endogenous human protein, annexin V (molecular weight approximately 35,000), has an affinity of about 10(-9) M for phosphatidylserine exposed on the surface of apoptotic cells. Annexin V can be labelled with radionuclides such as iodine or technetium, or positron emitting agents. Experimental studies in cells confirm that fluorescence and 99Tc(m)-labelled annexin have comparable affinity for apoptotic cells. In vivo studies with 99Tc(m)-labelled annexin confirm that radiolabelled annexin V can be used to image apoptotic cells/tissues in vivo. In this article, we review experimental data using annexin V imaging and discuss its possible future use to identify apoptosis in vivo.

Abstract

To assess the value of imaging rejection-induced apoptosis with technetium 99m and annexin V, a human protein-based radiopharmaceutical used in the diagnosis of acute rejection of a liver transplant, in a well-characterized rodent model of orthotopic liver transplantation.99mTc-radiolabeled annexin V was intravenously administered to six allografted (immunologically mismatched) and five isografted (immunologically matched) recipient rats on days 2, 4, and 7 after orthotopic liver transplantation. Animals were imaged 1 hour after injection of 0.2-2.0 mCi (8.0-74.0 MBq) of radiolabeled annexin V by use of clinical nuclear scintigraphic equipment.All animals in the allografted group demonstrated marked increases of 55% and 97% above the activity in the isografted group in hepatic uptake of annexin V on days 4 and 7, respectively. Severe acute rejection was histologically detected in all allografted livers on day 7. There was no histologic evidence of acute rejection in isografted animals. Dynamic hepatobiliary imaging with 99mTc and mebrofenin, an iminodiacetic acid derivative, demonstrated no correlation with the presence or absence of acute rejection or with annexin V uptake.Noninvasive imaging with radiolabeled annexin V is more sensitive and specific than imaging with 99mTc-mebrofenin in the diagnosis of acute rejection of a liver transplant.

Abstract

Apoptosis, also known as programmed cell death, is an indispensable component of normal human growth and development, immunoregulation and homeostasis. Apoptosis is nature's primary opponent of cell proliferation and growth. Strict coordination of these two phenomena is essential not only in normal physiology and regulation but in the prevention of disease. Programmed cell death causes susceptible cells to undergo a series of stereotypical enzymatic and morphologic changes governed by ubiquitous endogenous biologic machinery encoded by the human genome. Many of these changes can be readily exploited to create macroscopic images using existing technologies such as lipid proton magnetic resonance (MR) spectroscopy, diffusion-weighted MR imaging and radionuclide receptor imaging with radiolabeled annexin V. In this review the cellular phenomenon of apoptotic cell death and the imaging methods which can detect the process in vitro and in vivo are first discussed. Thereafter an outline is provided of the role of apoptosis in the pathophysiology of clinical disorders including stroke, neurodegenerative diseases, pulmonary inflammatory diseases, myocardial ischemia and inflammation, myelodysplastic disorders, organ transplantation, and oncology, in which imaging may play a critical role in diagnosis and patient management. Objective imaging markers of apoptosis may soon become measures of therapeutic success or failure in both current and future treatment paradigms. Since apoptosis is a major factor in many diseases, quantification and monitoring the process could become important in clinical decision making.

Abstract

Sonography, CT, and MR imaging are commonly used to screen for neonatal intracranial ischemia and hemorrhage, yet few studies have attempted to determine which imaging technique is best suited for this purpose. The goals of this study were to compare sonography with CT and MR imaging prospectively for the detection of intracranial ischemia or hemorrhage and to determine the prognostic value(s) of neuroimaging in neonates suspected of having hypoxic-ischemic injury (HII).Forty-seven neonates underwent CT (n = 26) or MR imaging (n = 24) or both (n = 3) within the first month of life for suspected HII. Sonography was performed according to research protocol within an average of 14.4 +/- 9.6 hours of CT or MR imaging. A kappa analysis of interobserver agreement was conducted using three independent observers. Infants underwent neurodevelopmental assessment at ages 2 months (n = 47) and 2 years (n = 26).CT and MR imaging had significantly higher interobserver agreement (P < .001) for cortical HII and germinal matrix hemorrhage (GMH) (Grades I and II) compared with sonography. MR imaging and CT revealed 25 instances of HII compared with 13 identified by sonography. MR imaging and CT also revealed 10 instances of intraparenchymal hemorrhage (>1 cm, including Grade IV GMH) compared with sonography, which depicted five. The negative predictive values of neuroimaging, irrespective of technique used, were 53.3% and 58.8% at the 2-month and 2-year follow-up examinations, respectively.CT and MR imaging have significantly better interobserver agreement for cortical HII and GMH/intraventricular hemorrhage and can reveal more instances of intraparenchymal hemorrhage compared with sonography. The absence of neuroimaging findings on sonograms, CT scans, or MR images does not rule out later neurologic dysfunction.

Abstract

Either inadequate or excessive apoptosis (programmed cell death) is associated with many diseases. A method to image apoptosis in vivo, rather than requiring histologic evaluation of tissue, could assist with therapeutic decision making in these disorders. Programmed cell death is associated with a well-choreographed series of events resulting in the cessation of normal cell function, and the ultimate disappearance of the cell. One component of apoptosis is signaling adjacent cells that this cell is committing suicide by externalizing phosphatidylserine to the outer leaflet of the cell membrane. Annexin V, a 32-kDa endogenous human protein, has a high affinity for membrane-bound phosphatidylserine. We have coupled annexin V with the bifunctional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m HYNIC-annexin V and demonstrated localization of radioactivity in tissues undergoing apoptosis in vivo. In this report we describe the results of a series of experiments in mice and rats to characterize the biologic behavior of (99m)Tc-HYNIC- annexin V. Biodistribution studies were performed in groups of rats at 10-180 min after intravenous injection of (99m)Tc-HYNIC-annexin V. In order to estimate the degree of apoptosis required for localization of (99m)Tc-annexin V in vivo, mice were treated with dexamethasone at doses ranging from 1 to 20 mg/kg, 5 h prior to (99m)Tc-HYNIC-annexin V administration, to induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin V injection; thymocytes were isolated, incubated with Hoechst 33342 followed by propidium iodide, and analyzed on a fluorescence-activated cell sorter. Each sorted cell population was counted in a scintillation counter. To test (99m)Tc-HYNIC-annexin V as a tracer for external radionuclide imaging of apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr/lpr mice) and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1 h post injection. Rat biodistribution studies demonstrated a blood clearance half-time of less than 10 min for (99m)Tc-HYNIC-annexin V. The kidneys had the highest concentration of radioactivity at all time points. Studies in the mouse thymus demonstrated a 40-fold increase in (99m)Tc-HYNIC-annexin V concentration in apoptotic thymocytes compared with the viable cell population. A correlation of r=0.78 was found between radioactivity and flow cytometric and histologic evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed a substantial increase of activity in the liver of wild-type mice treated with anti-Fas, while there was no significant change, irrespective of anti-Fas administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were obtained in wild-type mice 30 min after injection of (99m)Tc-HYNIC-annexin V. The imaging results were consistent with histologic analysis in these animals. In conlusion, these studies confirm the value of (99m)Tc-HYNIC-annexin V uptake as a marker for the detection and quantification of apoptotic cells in vivo.

Abstract

If life is to continue, cells that have completed their useful function(s) must die in a timely manner. Apoptosis, programmed cell death, is a natural, orderly, energy-dependent process that causes cells to die without inducing an inflammatory response. In the heart, apoptosis plays pivotal roles in the development of myocarditis, cardiomyopathies, transplant rejection, the periinfarct zone in myocardial infarction, and reperfusion injury. Apoptosis is triggered either by a decrease in factors required to maintain the cell in good health or by an increase in factors which cause damage to the cell. When these factors tilt in the direction of death and the cell has sufficient time to respond, a common proteolytic cascade involving cysteine aspartic acid-specific proteases (caspases) is activated to initiate apoptosis. Cells that die by apoptosis autodigest their DNA and nuclear proteins, change the phospholipid composition on the outer surface of their cell membrane, and form lipid enclosed vesicles, which contain noxious intracellular contents, organelles, autodigested cytoplasm, and DNA. The compositional cell membrane phospholipid change that occurs with the onset of apoptosis is marked by the sudden expression of phosphatidylserine (PS), a phospholipid that ordinarily appears on the inner leaflet of the membrane, on the external leaflet of the membrane. The constant exposure of PS during apoptosis makes it an attractive target for radiopharmaceutical imaging. An endogenous human protein, annexin V, has a high affinity (kd = 7 nmol/L) for PS bound to the cell membrane. Fluorescence-labeled annexin V is used for histologic and cell-sorting studies to identify apoptotic cells. Annexin has been radiolabeled and binds to cells undergoing apoptosis in vivo. This review outlines some of the key features of apoptosis as contrasted to necrosis (unregulated cell death) and describes how these processes can be imaged with radionuclide techniques.

Abstract

Programmed cell death, apoptosis, is an inducible, organized, energy requiring form of demise that results in the disappearance of a cell without the induction of an inflammatory response. Apoptotic cell death is strikingly different than necrotic death, which is disorderly, does not require energy and results in local inflammation, usually secondary to sudden release of intracellular contents. Apoptosis is induced when cells undergo severe injury to their nucleus, as occurs following exposure to gamma or X-radiation, or mitcochondria, as occurs in a variety of viral illnesses. Apoptosis can also be induced by external signals, such as interaction of fas ligand with fas receptors. Once the cell is committed to apoptosis, the caspase enzyme cascade is activated. An early effect of caspase activation is the rapid expression of phosphatidylserine on the external leaflet of the cell membrane. Membrane bound phosphatidylserine expression serves as a signal to surrounding cells, identifying the expressing cell as undergoing apoptosis. A deficiency or an excess of programmed cell death is an integral component of autoimmune disorders, transplant rejection and cancer. A technique to image programmed cell death would be useful to assist in the development of drugs designed to treat these diseases, and to monitor the effectiveness of therapy. The sudden expression of phosphatidylserine on the cell membrane is a target that could be used for this purpose. A 35 kD physiologic protein, Annexin V lipocortin, binds with nanomolar affinity to membrane bound phosphatidylserine. Annexin V has been radiolabeled with Technetium-99m by direct coupling to free sulfhydryl groups, and through the hydrazinonicatinamide and N2S2 linking agents. The biodistribution of the agents labeled with each of the methods is slightly different. In all cases the radiopharmaceutical binds to cells undergoing apoptosis in vitro, and permits imaging of the process in experimental animals.

Abstract

Apoptosis is a ubiquitous set of cellular processes by which superfluous or unwanted cells are eliminated in the body without harming adjacent healthy tissues. When apoptosis is inappropriate (too little or too much), a variety of human diseases can occur, including acute heart or lung transplant rejection.Our group has developed a new radiopharmaceutical, radiolabeled annexin V, which can image apoptosis.Here we briefly review the biomolecular basis of apoptosis and its role in acute rejection. We also describe the possible use of radiolabeled annexin V to screen children noninvasively for acute rejection following organ transplantation.

Abstract

Apoptosis (programmed cell death) is a critical element in normal physiology and in many disease processes. Phosphatidylserine (PS), one component of cell membrane phospholipids, is normally confined to the inner leaflet of the plasma membrane. Early in the course of apoptosis, this phospholipid is rapidly exposed on the cell's outer surface. Annexin V, an endogenous human protein, has a high affinity for membrane-bound PS. This protein has been labeled with fluorescein and has been used to detect apoptosis in vitro. We describe the use of radiolabeled annexin V to detect apoptosis in vivo. The results are compared to histologic and flow cytometric methods to identify cells and tissues undergoing apoptosis.Annexin V was coupled to hydrazinonicotinamide (HYNIC) and radiolabeled with 99mTc. Bioreactivity of 99mTc-HYNIC annexin V was compared with fluorescein isothiocyanate (FITC)-labeled annexin V in cultures of Jurkat T-cell lymphoblasts and in ex vivo thymic cell suspensions undergoing apoptosis in response to different stimuli. In addition, the uptake of FITC annexin V and 99mTc-HYNIC annexin V was studied in heat-treated necrotic Jurkat T-cell cultures. In vivo localization of annexin V was studied in Balb/c mice injected with 99mTc-HYNIC annexin V before and after induction of Fas-mediated hepatocyte apoptosis with intravenously administered antiFas antibody.Membrane-bound radiolabeled annexin V activity linearly correlated to total fluorescence as observed by FITC annexin V flow cytometry in Jurkat T-cell cultures induced to undergo apoptosis in response to growth factor deprivation (N = 10, r2 = 0.987), antiFas antibody (N = 8, r2 = 0.836) and doxorubicin (N = 10, r2 = 0.804); and in ex vivo experiments on thymic cell suspensions with dexamethasone-induced apoptosis from Balb/c mice (N = 6, r2 = 0.989). Necrotic Jurkat T-cell cultures also demonstrated marked increases in radiopharmaceutical (4000-5000-fold) above control values. AntiFas antibody-treated Balb/c mice (N = 6) demonstrated a three-fold rise in hepatic uptake of annexin V (P < 0.0005) above control (N = 10), identified both by imaging and scintillation well counting. The increase in hepatic uptake in antiFas antibody-treated mice correlated to histologic evidence of fulminant hepatic apoptosis.These data suggest that 99mTc-HYNIC annexin V can be used to image apoptotic and necrotic cell death in vivo.

Abstract

Apoptosis, or programmed cell death, has been suggested as a mechanism of immunologic injury during cardiac allograft rejection. We tested the hypothesis that technetium Tc 99m annexin V, a novel radiopharmaceutical used to detect apoptosis, can be used to detect cardiac allograft rejection by nuclear imaging.Untreated ACI rats served as recipients of allogeneic PVG rat (n = 66) or syngeneic ACI rat (n = 30) cardiac grafts. Untreated recipient animals underwent 99mTc-annexin V imaging daily for 7 days. Region of interest analysis was used to quantify the uptake of 99mTc-annexin V. Immediately after imaging grafts were procured for histopathologic analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling of apoptotic nuclei. One group was treated with 10 mg/kg/d cyclosporine (INN: ciclosporin) commencing on day 4 after transplantation (n = 6).Untreated allografts showed histologic signs of rejection 4 days after transplantation. Apoptotic nuclei could be demonstrated in myocytes, endothelial cells, and graft-infiltrating cells of all rejecting allografts. Nuclear imaging revealed a significantly greater uptake of 99mTc-annexin V in rejecting allogeneic grafts than in syngeneic grafts on day 4 (P = .05), day 5 (P < .001), day 6 (P < .001), and day 7 (P = .013) after transplantation. A correlation between the histologic grade of acute rejection and uptake of 99mTc-annexin V was observed (r2 = 0.87). After treatment of rejection with cyclosporine, no apoptotic nuclei could be identified in allografts and uptake of 99mTc-annexin V decreased to baseline.Apoptosis occurs during acute cardiac allograft rejection and disappears after treatment of rejection. 99mTc-annexin V can be used to detect and monitor cardiac allograft rejection.

Abstract

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.

Abstract

To evaluate regional cerebral blood flow with power and pulsed wave Doppler ultrasound (US) in extremely low-birth-weight neonates with periventricular leukomalacia (PVL), germinal matrix hemorrhage (GMH), or both.The lenticulostriate arteries of 17 preterm neonates (birth weight < or = 1,100 g) were assessed daily with Doppler US during the first 5-6 days of life. The mean arterial pressure and bilateral peak velocity, resistive index, coronal vascular cross-sectional area, and product of the peak velocity and vascular cross-sectional area were measured.Five neonates developed PVL, GMH, or both; results of follow-up examinations in 11 patients were normal. One neonate with severe intrauterine growth retardation and renal tubular acidosis was excluded. Neonates with PVL, GMH, or both showed significantly greater mean values and more variable values of vascular cross-sectional area and product of peak velocity and cross-sectional area than neonates without PVL or GMH (P < .025). Mean resistive index was significantly lower in neonates with PVL, GMH, or both than in neonates without (P < .01). There were no significant differences between mean arterial pressure in neonates with and those without PVL, GMH, or both.By enabling the detection of autoregulatory fluctuations in cerebral blood flow associated with hypoxic-ischemic injury, power and pulsed wave Doppler US may enable identification of preterm neonates who are at risk of developing PVL, GMH, or both during the 1st week of life.

Abstract

Quantification of apoptotic cell death in vivo has become an important area of investigation in patients with acute lymphoblastic leukemia (ALL). We have devised a noninvasive analytical method to estimate the percentage of apoptotic lymphoblasts in doxorubicin-treated Jurkat T-cell ALL cultures, using proton nuclear magnetic resonance spectroscopy (1H NMR). We have found that the ratio of the methylene (CH2) resonance (at 1.3 ppm) to the methyl (CH3) resonance (at 0.9 ppm) signal intensity, as observed by 1H NMR, is directly proportional to the percentage of apoptotic lymphoblasts in vitro. The correlation between the CH2/CH3 signal intensity ratio and the percentage of apoptotic lymphoblasts was optimal 24 to 28 hours after doxorubicin treatment (r2 = .947, N = 27 samples). There was also a direct temporal relationship between an increase in the CH2/CH3 signal intensity ratio and the onset of apoptosis as detected by nuclear morphologic analysis, fluorescein-annexin V flow cytometry, and DNA gel electrophoresis. Thin-layer chromatography confirmed that a dynamic and/or compositional change of the plasma membrane, rather than increases in lipase activity or fatty acid production, appears to account for the increase in the CH2/CH3 signal intensity ratio during apoptosis. 1H NMR may have clinical utility for the early noninvasive assessment of chemotherapeutic efficacy in patients with ALL.

Abstract

To assess the usefulness of ultrasound (US), computed tomography (CT), and magnetic resonance (MR) imaging in the detection of intracranial hemorrhage and ischemia in newborns.Seventy-six neonates who underwent US within 72 hours of CT or MR examination were studied. Four observers rated images for the presence of germinal matrix hemorrhage (GMH), intraventricular hemorrhage (IPH), extraaxial hemorrhage, and hypoxic-ischemic encephalopathy.In 39% of neonates, CT and MR imaging provided greater confidence than US for the diagnosis or exlusion of neonatal ischemia or hemorrhage. Kappa analysis revealed significantly better interobserver agreement with CT than with US for the detection of GMH, IVH, IPH, and cortical infarction or ischemia (P

Abstract

Cells undergoing apoptosis (programmed cell death) display profound morphologic and biochemical changes in the nucleus, cytoplasm, and plasma membrane. We have shown a direct temporal relationship between the onset of apoptosis in Jurkat T-cell lymphoblast cultures and a greater than two-fold increase in the signal intensity of the methylene resonance (at 1.3 ppm) as observed by proton nuclear magnetic resonance spectroscopy (1H NMR). The increase in the methylene resonance intensity was seen when apoptosis was induced by serum deprivation, glucocorticoid, and doxorubicin treatment but not in necrotic (nonapoptotic) cell death. We have found similar changes in a variety of other cell lines undergoing apoptosis including the Hut 78 T-cell leukemia, JY natural killer T-cell leukemia, Daudi B-cell lymphoma, HeLa, and 3T3 fibroblast cell lines. Furthermore, this spectral change was diminished in Bcl-2 overexpressing HL-60 cell cultures treated with doxorubicin, which were relatively resistant to apoptosis, as compared to apoptotic HL-60 cultures. 1H NMR spectroscopy therefore may be useful in detecting apoptotic cell death in vivo.

Abstract

The most frequently occurring and important cause of gastric outlet obstruction in the neonate and young infant is infantile hypertrophic pyloric stenosis (IHPS). A reported association of IHPS and eosinophilic gastroenteritis [1] raises interesting questions about the possible etiologic relationship between the two entities. It is plausible that the observed sonographic pyloric muscular wall thickness in IHPS may in part be dependent on the degree and duration of an allergic gastroenteropathy. A recent report suggests that endoscopy may be a more reliable diagnostic method than sonography in the patient with evolving IHPS [2]. Our recent experience with a patient with evolving IHPS supports the findings described in these prior reports.

Abstract

To improve the prediction of individual survival in patients with intracranial astrocytomas through the analysis of volumetric tumor doubling time (VDt) and DNA ploidy.A pilot study was retrospectively conducted on a group of 25 patients with intracranial astrocytomas in whom recurrent and/or progressive disease was observed on serial contrast-enhanced CT or MR examinations. VDt was computed using two or more data points from a semilogarithmic plot of tumor volume versus time. Size-adjusted survival was calculated using a method based on VDt and initial tumor volume to decrease the lead time bias attributable to differing tumor sizes at presentation.Slower VDt was associated with significantly longer survival and size-adjusted survival as determined by a univariate Cox proportional hazard analysis. Aneuploidy was a significant indicator of poor survival. Aneuploid and multiclonal astrocytomas had poor size-adjusted survivals compared with diploid astrocytomas. Grade IV astrocytomas had significantly poorer survival and size-adjusted survival compared with lower grades (I to III), which individually were not significantly correlated. However, grade IV histology was not a significant independent predictor of size-adjusted survival in a multivariate Cox model, whereas VDt and DNA ploidy remained significant. VDt also had a significant direct linear correlation to survival and size-adjusted survival.VDt and DNA ploidy were more sensitive than histologic grading as indicators of individual survival. Initial tumor size needs to be considered when staging and assessing survival in patients with intracranial astrocytomas.

Abstract

A new experimental technique is described that allows the characterization of the angle-dependent ultrasonic scattering properties of isolated breast tissue lobules. A review of breast tissue micro-architecture is presented as background material. Measured estimates of the scatter angle-dependent differential scatter cross-sections (DSC) from 31 excised lobules (14 cancer in situ, 17 noncancer) were examined, and dominant trends described by statistical factors. Three factors were extracted, using principal component factor analysis, which collectively accounted for over 70% of the scatter angle-dependent variation exhibited by the measured data.

Abstract

Normalized estimates of the scattering angle-dependent differential scattering cross-section (DSC) at 1.0 MHz were measured from 278 samples of excised breast tissue taken from 66 women. A comparison of results for samples that contained tissue structures previously associated with an increased probability of developing breast cancer to those that did not contain high-risk structures showed that the average magnitude of DSC estimates was insufficient to identify samples with high-risk lesions. Principal component factor analysis (PCFA) was applied to extract scattering angle-dependent trends common to the entire data base. The normalized estimates of the measured DSCs (NDSC) from tissue samples are compared to estimates previously obtained from isolated breast tissue lobules as well as with results from the PCFA. Results are presented that indicate that the dominant angle-dependent trends in the NDSC results are independent of the age of the patient and are similar to trends extracted from isolated breast tissue lobules. The breast tissue structure common to all of these specimens is the terminal duct.

Abstract

A murine model of implanted CNS neoplasia was used to study a new form of brain tumor immunotherapy with intralesional Corynebacterium parvum (C. parvum). Assessment of treatment protocols has been limited by the inability to assess, noninvasively, tumor burden and/or the inflammatory reaction induced in the murine brain by treatment with C. parvum. This study demonstrates that contrast-enhanced MR imaging can monitor in vivo tumor burden and the immune response to intracerebral C. parvum. KHT murine sarcoma was stereotaxically implanted into the right frontal lobe of C3H/HeN mice at doses of 10,000 and 50,000 tumor cells. The KHT sarcoma is 100% fatal in untreated mice. Therapy consisted of an intraperitoneal injection of 350 micrograms of killed C. parvum 1 day after tumor implantation followed by 70 micrograms of C. parvum stereotaxically injected into the tumor 5 days after implantation. MR imaging was performed on mice injected with saline only, C parvum only, tumor only, and tumor treated with C. parvum. C. parvum alone elicited an intense transitory mononuclear cell inflammatory reaction in the meninges, ependyma, and to a variable degree at the injection site. The inflammatory response reached a peak 2 weeks after intracerebral injection. Contrast-enhanced MR imaging was able to detect the presence and severity of C. parvum-induced inflammation, which decreased 3 weeks after intracerebral injection. The transitory nature of this type of inflammation should allow its differentiation from tumor in subjects undergoing serial scanning following intracerebral injection of C. parvum as a form of brain tumor immunotherapy.

Abstract

The sonographic and computed tomographic (CT) findings were reviewed in 17 patients with acute acalculous cholecystitis (AAC) over a 6-year period from 1984 to 1989. Of the six patients in whom both ultrasound and CT were performed, CT revealed marked gallbladder (GB) wall abnormalities, including perforation, and pericholecystic fluid collections in five patients not demonstrated by sonography. Of the total group, five patients had GB wall thicknesses of less than or equal to 3 mm (normal) at pathologic examination, which demonstrated a spectrum of disease ranging from acute hemorrhagic/necrotizing, to gangrenous acalculous cholecystitis with perforation. Sonography was falsely negative or significantly underestimated the severity of AAC in seven of the 13 patients examined by sonography. CT because of its superior ability to assess pericholecystic inflammation may provide additional diagnostic information even after a thorough sonographic study in cases of AAC.

Abstract

A pilot study involving 53 specimens of excised human female breast tissue was performed to provide preliminary estimates of the acoustic shear wave speed of sound and linear attenuation coefficient associated with high risk for development of invasive breast cancer. The measured shear wave properties were studied as a function of: 1. whether the tissue was presented as a 2.0 to 4.0 gram mass biopsy sample or a 2.0 to 4.0 gram mass subsample taken from a larger mastectomy source; 2. the time post-excision prior to shear wave characterization; 3. the risk factor associated with the number and types of lesions found in the specimen; and 4. the percent by volume of fat or collagen present in the sample. The shear speed of sound was found to range between 20 and 900 m/s with a tendency for lower speeds as a function of time post excision. The results for shear attenuation coefficients ranged from 300 to 9000 cm-1 and did not show a marked time dependence. Average results from mastectomy specimens differed from those for biopsies, but the difference may have been due to variations in tissue composition and time post excision before shear wave characterization.